Multifunctional recombinant phycobiliprotein-based fluorescent constructs and phycobilisome display

ABSTRACT

The invention provides multifunctional fusion constructs which are rapidly incorporated into a macromolecular structure such as a phycobilisome such that the fusion proteins are separated from one another and unable to self-associate. The invention provides methods and compositions for displaying a functional polypeptide domain on an oligomeric phycobiliprotein, including fusion proteins comprising a functional displayed domain and a functional phycobiliprotein domain incorporated in a functional oligomeric phycobiliprotein. The fusion proteins provide novel specific labeling reagents.

The research carried out in the subject application was supported inpart by grants from the Department of Energy (Grant No.RDE-FG-91ER61125). The government may have rights in this invention.

FIELD OF THE INVENTION

The field of the invention is phycobiliprotein-assisted expression andfolding of proteins and the production of function-added recombinantphycobiliproteins.

BACKGROUND OF THE INVENTION

Many foreign proteins expressed in bacteria form insoluble aggregatescalled inclusion bodies. Recovery of the recombinant protein of interestrequires dissolving the inclusion bodies under denaturing conditions,removing the denaturant to allow the recombinant protein to fold, andfinally purifying the recombinant protein. This process is laborious andfrequently gives low yields. Inclusion bodies are formed because theaggregation of the newly synthesized recombinant polypeptide is fasterthan its folding into the native structure. In one aspect, the inventionprovides bifunctional fusion constructs which are rapidly incorporatedinto a macromolecular structure such that the fusion proteins areseparated from one another and unable to self-associate.

In a more particular aspect, the macromolecular structures areoligomeric phycobiliproteins—a family of structurally relatedphotosynthetic accessory proteins naturally found, inter alia, incyanobacteria, the chloroplasts of the Rhodophyta (red algae) and inthose of the Cryptophyceae. When they carry covalently attached lineartetrapyrrole prosthetic groups (bilins), these proteins can exhibitbrilliant colors and intense fluorescence, making them valuable specificlabeling reagents. Unfortunately, the diversity of such reagents hasbeen restricted to naturally occurring phycobiliproteins and theirtarget specificity generated by chemical conjugation, which generatesheterogeneity (see, e.g. Siiman et al., 1999, Bioconj Chem, 10,1090-1106). The use of phycobiliproteins in the invention also overcomesthese prior art limitations and provides homogenous, specific labelingreagents.

SUMMARY OF THE INVENTION

We have found that phycobiliprotein subunits are tolerant to terminalextensions of different sizes. A phycobiliprotein subunit domain in afusion protein is able to assemble with a cognate partner subunit toform heterodimers, and frequently further assemble into evenhigher-order aggregates, and into the light-harvesting antennacomplexes, phycobilisomes. The non-phycobiliprotein part of the fusionprotein (the displayed domain) is exposed on the surface of theoligomeric phycobiliproteins. We define this fusion protein expressionsystem as “phycobilisome display”. Newly synthesized fusion polypeptidesare quickly separated from each other by virtue of the assembly of thephycobiliprotein domain (the carrier domain) into oligomers. Thenon-phycobiliprotein moiety (the displayed domain) of the fusion proteinis displayed on the surface of oligomer, e.g. the rods of thephycobilisome, and is able to fold into functional proteins whilesequestered from interaction with other unfolded polypeptides.Phycobilisome display can therefore be used as an alternative foldingenvironment for difficult-to-fold proteins, especially those that havebeen found difficult to fold in other organisms. The displayed proteincan be used as a fusion construct bearing a fluorescent phycobiliproteintag, or be separated from the carrier phycobiliprotein domain bycleavage of the linker peptide between the carrier domain and thedisplayed domain by a specific protease. Either embodiment may bepracticed in cells (e.g. using a resident protease) or in vitro (e.g.using purified fusion proteins, cell-free expression systems, etc.),though cleavage is preferably practiced outside the cell to control itstiming with respect to folding.

Accordingly, the invention provides methods and compositions fordisplaying a functional polypeptide domain on an oligomericphycobiliprotein. The compositions include fusion proteins comprising afunctional displayed domain and a functional phycobiliprotein domainincorporated in a functional oligomeric phycobiliprotein. In particularembodiments, the phycobiliprotein domain is a natural phycobiliproteindomain or modified variant thereof; the functional oligomericphycobiliprotein is an α, β heterodimer; the displayed domain comprisesan affinity tag, an oligomerization moiety, a specific binding moietyand/or a signaling moiety; and/or the displayed domain is refractive toexpression in E. coli. The compositions also include functionaloligomeric phycobiliproteins comprising the subject fusion proteins andcells comprising such oligomeric phycobiliproteins.

The subject methods include methods for making a functional displayeddomain comprising the step of combining a polypeptide comprising adisplayed domain and a phycobiliprotein domain with a phycobiliproteinsubunit under conditions to form a subject fusion protein. In particularembodiments, the methods further comprise prior to the combining step,the step of making the polypeptide by expressing a nucleic acid encodingthe polypeptide; and/or after the combining step, the step of separatingthe functional displayed domain from the functional phycobiliproteindomain. The methods steps may occur intracellularly, e.g. in a cellwhich is or is a progeny of a natural cell which naturally makesfunctional phycobiliprotein, or a cell engineered to produce functionalphycobiliprotein.

DETAILED DESCRIPTION OF PARTICULAR EMBODIMENTS OF THE INVENTION

The following descriptions of particular embodiments and examples areoffered by way of illustration and not by way of limitation.

The invention provides methods and compositions for displaying afunctional polypeptide (the foreign protein or displayed domain) on anoligomeric phycobiliprotein. The compositions include fusion proteinscomprising a functional displayed domain and a functionalphycobiliprotein domain incorporated in a functional oligomericphycobiliprotein.

A functional phycobiliprotein domain is capable of assembling the fusionprotein in a functional oligomeric phycobiliprotein. Preferred domainsprovide at least 1%, preferably at least 10%, more preferably at least50%, more preferably at least 75%, more preferably at least 90% and mostpreferably substantially equivalent oligomer assembly ability as that ofa corresponding unfused phycobiliprotein, as measured in competitionassays, e.g. as described herein.

Any phycobiliprotein domain having the requisite functionality may beused and these may be derived from natural, semisynthetic or syntheticsequences. A wide variety of natural phycobiliproteins are known in theart, e.g. Apt and Grossman, 1995, J Mol Biol 248, 79-96, includingproteins derived from many cyanobacteria, rhodophytes (red algae) andcryptomonads, etc. (see, e.g. Glazer, 1994, J Appl Phycol 6, 105-112;Glazer et al. 1995, Photosynth Res 46, 93-105), particularlyphycoerythrins, phycocyanins, and allophycocyanins. In addition, a widevariety of methods are known for modifying such natural sequences togenerate semi-synthetics, e.g. Glazer, 1994, supra, describesphycobiliproteins having non-natural, predetermined bilin compositions,and Toole et al., 1998, Mol Micro 30, 475-486 describes recombinationsof phycobiliprotein deletion mutants. Finally, known phycobiliproteinstructure-function relationships (e.g. Anderson et al., 1998, Mol Micro30, 467-474) are exploited to generate synthetic sequence analogs usingconventional methods.

In a particular embodiment, the phycobiliprotein domain ischaracterizable as an α or β subunit, based on its sequence similarityto natural α and β phycobiliprotein subunits. The selection of α or βphycobiliprotein domains may yield different results, affecting thedisplayed domain, the carrier protein, or both, and such differencesguide the selection of the carrier phycobiliprotein subunit. Forexample, the phycocyanin α subunit and the β-L11-α subunit-fusion arepreferred when the foreign protein is displayed on the N-terminus of thecarrier protein because of the higher yield and the better spectroscopicproperties of the resulting fusion protein. For display on theC-terminus, phycocyanin β subunit and the α-L11-β subunit fusion arebetter suited because the phycocyanin a subunit is sensitive toextension on its C-terminus (usually leading to incomplete bilinaddition and in some instances partial unfolding of the protein).

In a particular embodiment, the phycobiliprotein domain confersfluorescence on the fusion protein, preferably providing fluorescencequantum yield and molar extinction coefficients at least 1%, preferablyat least 10%, more preferably at least 50%, more preferably at least75%, more preferably at least 90% and most preferably substantiallyequivalent to that of a corresponding unfused phycobiliprotein, measuredas described herein. Preferred domains provide extinction coefficientsof at least 100K, preferably at least 300K, more preferably at least 1Mand/or quantum yields of at least 0.25, preferably at least 0.5, morepreferably at least 0.6, as described herein. In other preferred aspectsof this embodiment, the fluorescence emission spectrum of the fusionprotein is substantially equivalent to that of a corresponding unfusedphycobiliprotein.

In a particular embodiment, the phycobiliprotein domain of the fusionprotein comprises one or more bilins, preferably functional bilinscontributing to the visible absorption spectrum of the phycobiliproteindomain and fusion protein, preferably natural bilins. In a particularembodiment, the bilins are covalently coupled to the phycobiliproteindomain, preferably through cysteine thioether linkages, preferably atnatural bilin attachment sites. The bilins may be coupled to thephycobiliprotein domain by any mechanism that provides the requisitefunctionalities, including enzymatic addition. Accordingly, in aparticular embodiment, the phycobiliprotein domain of the fusion proteinprovides a substrate for enzymatic bilin addition, which may providenatural or non-natural bound bilin distribution, preferably a substratefor enzymes which naturally modify a corresponding naturalphycobiliprotein.

The displayed domain may be any polypeptide having a requisitefunctionality and being compatible with that of the phycobiliproteindomain and with assembly of the fusion protein in a functionaloligomeric phycobiliprotein. Accordingly, a wide range of displayeddomains may be used including domains comprising an affinity tag, anoligomerization moiety, a specific binding moiety and/or a signalingmoiety, and including polypeptides which may be coupled tophycobiliproteins by chemical conjugation invention (see, inter alia,U.S. Pat. Nos. 4,520,110; 4,542,104; 4,857,474; 5,055,556; 5,707,804;5,728,528; and 5,869,255).

The displayed domain is preferably of length and sequence sufficient toprovide a constrained structure, including at least secondary structure,preferably tertiary structure. In a particular embodiment, theconstrained structure is of complexity sufficient to require complexfolding and is poorly expressed independent of the fusion protein inactive form in conventional expression systems, particularlyconventional yeast (e.g. S. cerevisiae) and bacterial (e.g. E. coli)expression systems. Preferred displayed domains are refractive toexpression when expressed independent of the fusion proteins, i.e.substantially not expressed in active form, in conventional expressionsystems, particularly E. coli. The displayed domain may have anyspectrophotometric properties compatible with the requisitefunctionalities of the fusion protein. In a particular embodiment, thedisplayed domain is substantially transparent to the wavelengths ofvisible light absorbed by phycobiliproteins, and therefore does notsubstantially affect the fusion protein and/or oligomericphycobiliprotein light-harvesting function, and/or is substantiallytransparent to the wavelength(s) of energy emitted by thephycobiliprotein domain, and therefore is not substantially affected bysuch energy.

The displayed domain may frequently be displayed on either terminus ofthe phycobiliprotein domain—important because many proteinspreferentially tolerate extension on one of their termini. Preferredexpression orientation is readily determined empirically, and in manycases (e.g. for making phycobiliprotein-labeled fluorescent reagents) isadvised by published C- and N-terminal GFP fusions. In particularembodiments, display on the N-terminus of a phycobiliprotein β subunit,rather than the α subunit, is more conducive to folding of certaindisplayed proteins. Use of the subunit-fusion phycocyanins α-L11-β andβ-L11-α as the carrier protein may also have certain advantages: thefusion proteins tend to be more stable (see, e.g. Example B, below), andthe 1:1 stoichiometry of α and β subunits is ensured.

The fusion proteins may comprise additional components as desired, whichmay provide modules of functionalities, such as affinity handles, dimer-or oligomerization domains, stabilization domains, specificity domains,signaling domains, etc., apart from any such functionality/ies providedby the displayed domain. For example, for constructs to be used asfluorescent labels, introduction of GCN oligomerization domains enhancesboth the spectroscopic value (more chromophores) and binding affinity(more sites for intermolecular interaction).

In particular embodiments, the fusion protein comprises a specificbinding moiety comprising at least one of a specific binding pair, suchas a receptor—ligand pair, e.g. an immunoglobulin antigen-binding domainor antigenic domain, a lectin saccharide-binding domain or glycosylatedor glycosylatable domain, an avidin or streptavidin biotin-bindingdomain or biotinylated or biotinylatable (i.e. providing a substrate forenzymatic biotinylation) domain, etc. In a particular embodiment, theprotein comprises a biotinylated or biotinylatable domain, which ispreferably biotinylated in the expression system (e.g. cell) selectedfor expression of the fusion protein. A wide variety of synthetic,semi-synthetic and natural such domains are known in the art, see e.g.,Schatz et al. 1993, Bio/Technology 11, 1138-1143; Tatsumi et al., 1996,Anal Biochem 243, 176-180; Samols et al. 1988, J Biol Chem 263,6461-6464, including homologs in phycobiliprotein producingcyanobacteria, e.g. Gornicki et al. 1993, J Bacteriol 175, 5268-5272;Phung et al., GenBank Accession No. U59235; Nakamura et al. 1998 NuclAcids Res 26, 63-67. In fact, enzymes sufficient to biotinylatebiotinylatable domains have been characterized (e.g. Beckett et al.1999, Protein Sci 8, 921-929; Buoncristiani et a). 1988, J Biol Chem263, 1013-1016), permitting in vitro biotinylation (e.g. Li et al.,1992, J Biol Chem 267, 855-863). These biotinylated domains permitespecially convenient affinity purification tags (e.g. Cronan 1990, JBiol Chem 265, 10327-10333, Example C, below) and are useful in the manywell developed biotin/avidin applications (e.g. Wilchek and Bayer (ed)1990, Methods Enzymol 184, Academic Press, NY).

In another example, various spacers or flexible linker peptidesproviding a variety of functionalities, such as a specific endopeptidaserecognition and/or cleavage site, an affinity-purification tag, etc.,may be used between the displayed and phycobiliprotein domains. Forexample, when displayed C-terminally to the phycobiliprotein domain, aspecific protease recognition and cleavage site can be engineeredimmediately upstream from the displayed domain so, upon cleavage withthe protease, the displayed domain can be cleanly released from thecarrier protein. This strategy also works for most proteins displayed onthe N-terminus of the carrier protein because the functions of mostdisplayed proteins are not affected by C-terminal extensions severalresidues long. In situations where such C-terminal extension is highlyundesirable, an intein domain (Perler F B, Jan. 1, 2000, Nucleic AcidsRes 28, 344-345 “InBase, the Intein Database”) can be engineeredimmediately downstream from the displayed domain. Subsequent excision ofintein cleanly releases the displayed domain from the carrier protein.

The linkers may also be used to facilitate display of domains that wouldotherwise interfere with oligomeric phycobilisome assembly. The lengthand amino acid sequence requirements of such functionality are readilydetermined empirically for a given fusion construct. Generally, thelinkers are preferably from at least 5, preferably at least 10 residuesin length, typically requiring no more than 50, and more often no morethan 30 residues. To facilitate an unintrusive orientation (see, e.g.Example A, below) small, flexible residues such as Ala, Gly and Ser areparticularly convenient components.

The fusion proteins are incorporated in a functional oligomericphycobiliprotein, see e.g. Glazer, 1994, Adv Mol Cell Biol 10, 119-149,comprising at least a dimer, preferably an α, β heterodimer. Theoligomers, especially when assembled into higher-order structures suchas trimers, hexamers, rods and phycobilisomes, constrain one displayedprotein from interacting with another. This is particularly useful inproducing proteins whose function is (1) harmful to the cell but (2)dependent on the formation of dimer or multimer. In a particularembodiment, the oligomeric fusion phycobiliproteins are structurallysubstantially identical to those of corresponding naturalphycobiliproteins and preferably provide fluorescence quantum yield andmolar extinction coefficients at least 1%, preferably at least 10%, morepreferably at least 50%, more preferably at least 75%, more preferablyat least 90% and most preferably substantially equivalent to those ofcorresponding natural phycobiliproteins. In other preferred aspects ofthis embodiment, the fluorescence emission spectrum of the oligomericfusion phycobiliproteins is substantially equivalent to those ofcorresponding natural phycobiliproteins.

Because higher order oligomers (phycobilisomes) can form a complex withthe water-splitting oxygen-evolving photosystem II, the surroundingenvironment of phycobilisomes, unlike virtually all other cellularenvironments, may be oxidative enough for spontaneous formation ofdisulfide bonds. Therefore phycobilisome display may be applied to theexpression and folding of disulfide bond-containing proteins.Accordingly, the compositions also include functional oligomericphycobiliproteins comprising the subject fusion proteins and cellscomprising such oligomeric phycobiliproteins.

The fusion proteins are expressible in any convenient system compatiblewith expression of the fusion protein, preferably an empiricallyoptimized host cell, host cell of the most closely related naturalphycobiliprotein, or lysate or extract thereof. In a particularembodiment, the fusion protein is expressed at least 1%, preferably atleast 10%, more preferably at least 50%, more preferably at least 75%,more preferably at least 90% and most preferably substantiallyequivalent to that of a corresponding unfused phycobiliprotein domain. Awide variety of expression systems may be used. For example, Anabaenacells expressing phycobilisome displayed proteins have normal phenotypeand growth rates and can be grown large scale in defined inorganic saltmedia. Alternatively, cells or protoplasts may be engineered orreconstituted to express requisite lyase subunits (e.g. cpcE and cpcF,e.g. Fairchild et al., 1994, J Biol Chem 267, 16146-16154) and/orphycobiliprotein linker proteins (e.g. cpcC, e.g. Swanson et al., 1992,J Bact 174, 2640-2647) under conditions wherein the requisite oligomericphycobilisome proteins are formed. Reconstituted heterologous cellularsystems require either high levels of phycobiliprotein domain expression(sufficient to form dimers; see, Glazer et al., 1973, J. Biol Chem 248,5679-5685) or the expression of a compatible heterophycobiliproteindomain (to form heterodimers), i.e. simply attempting to express alabeled phycobiliprotein fusion protein with a coexpressed lyase in aheterologous cell is insufficient to form the required oligomericphycobiliproteins (Schroeder 1997, Phycobiliproteins: Biosynthesis andApplications, UC Berkeley, Dissertation). Alternatively, extracellularor cell free systems, such as lysates, extracts and reconstituted invitro bilin addition systems may be used (e.g. Arciero et al. 1988, JBiol Chem 263; 18343-18349; 18350-18357; 18358-18363). Becausephycobiliproteins and phycobilisomes are made at very high levels in thecell, especially under low light intensity, phycobiliprotein displayalso helps to increase the yield of the displayed protein.

The subject methods include methods for making a functional displayeddomain, the method comprising the step of combining a polypeptidecomprising a displayed domain and a phycobiliprotein domain with aphycobiliprotein subunit under conditions to form a subject fusionprotein. In particular embodiments, the methods further comprise priorto the combining step, the step of making the polypeptide by expressinga nucleic acid encoding the polypeptide; and/or after the combiningstep, the step of separating the functional displayed domain from thefunctional phycobiliprotein domain. The methods steps may occurintracellularly, e.g. in a cell which is or is a progeny of a naturalcell which naturally makes functional phycobiliprotein.

Our extensive studies on phycobiliprotein subunit fusion constructsdemonstrate the versatility of phycobilisome display. In fact, ourdisplay concept may be practiced with other macromolecules, such asribosomes, and such macromolecular complex display can also help inposttranslational modification of proteins. For example, proteinsdisplayed on 80S ribosomes in eukaryotic cytosol can undergo differentposttranslational modifications than proteins displayed on 70S ribosomesin the mitochondria and chloroplasts. Similarly, proteins displayed ondifferent sides of the ER are also subjected to different modifications.

EXAMPLES AND DETAILED EXPERIMENTAL PROTOCOLS Example A. Expression andCharacterization of Recombinant Phycobiliprotein Fusion Proteins

We describe here the expression of genes engineered to encode Anabaenasp. PCC7120 C-phycocyanin α and β polypeptides bearing a 24-residueN-terminal peptide tag, incorporating a block of six His residues(generally referred to as the “6×His tag”), in wild-type and mutantcells of the filamentous cyanobacterium Anabaena sp. PCC7120. The 6×Histag allows one-step purification of the recombinant polypeptides byimmobilized metal-ion affinity chromatography [IMAC; (8)] away from thewild-type phycobiliproteins. We show here that phycocyanobilin (PCB) iscorrectly attached to the His-tagged phycocyanin α and β polypeptidesand that they fold to yield molecules whose spectroscopic propertiescorrespond to those of wild-type α and β polypeptides.

Cultures and Strains—Escherichia coli strain DH5α (11) grown in LBmedium was used in all cloning and expression experiments.Plasmid-encoded resistance to ampicillin or spectinomycin (Sp) wasselected at an antibiotic concentration of 100 μg ml⁻¹. Foroverexpression of His-tagged proteins, isopropyl-β-D-thiogalactoside(IPTG) was added to a final concentration of 0.5 mM to exponentiallygrowing cultures to derepress the trc promoter. Induced cultures wereusually grown with vigorous aeration for 5 hr at 30° C. to reduceformation of inclusion bodies. Induced cells were pelleted and stored at−20° C. until use.

Anabaena sp. strain PCC7120 and its derivative strains were grown at 30°C. on modified AA minimal medium plus nitrate (12) under medium (75 μEm⁻² s⁻¹) to high (200 μE m⁻² s⁻¹) light intensity provided by cool-whitefluorescent bulbs. Anabaena cultures overexpressing His-taggedphycocyanin subunits were grown in half-strength AA plus nitrate medium(bubbled with 5% CO₂ in air, with 2.5 mM HEPES pH 9.0 buffer to maintainpH) to late-exponential phase before addition of IPTG to 0.5 mM. Thecultures were induced for 2 to 3 days before cells were harvested andstored at −20° C. Triparental mating to introduce free-replicatingplasmids into Anabaena cells from E. coli was performed essentially asdescribed (13). Strains bearing the plasmid-borne aadA gene, encodingresistance to streptomycin (Sm) and to Sp, were selected at 1 μg ml⁻¹ Smplus 10 μg ml⁻¹ Sp on agar-solidified medium and at 20 μg ml⁻¹ Sp alonein liquid medium. Mutant strains B646, B64328, and B64407 generated bytransposon insertion (7) were selected with neomycin sulfate (Nm) at 200μg ml⁻¹ on solid medium and 10 μg ml⁻¹ in liquid medium.

Cloning of Anabaena sp. PCC7120 C-Phycocyanin α and β subunits—Standardprocedures were used for most molecular biological manipulations (14).Genomic DNA was isolated from Anabaena sp. PCC 7120 as described (15).The cpcA and cpcB genes encoding the α and β subunits of phycocyanin,respectively, were amplified for genomic DNA by the polymerase chainreaction (PCR; 16). The 0.5-kb PCR fragment was digested with therestriction enzymes NdeI and HindIII, and cloned into NdeI- andHindIII-digested cloning vector pUC19 [(17); GenBank accession No.X02514], giving plasmid pBS1385. The 0.5-kb PCR fragment was digestedwith enzymes NdeI and EcoRI, and cloned into NdeI- and EcoRI-digestedpUC19, giving plasmid pBS251. All fragments generated by PCR weresequenced to verify fidelity using the PRISM 373 DNA sequencing systemand the dye-terminator cycle-sequencing kit from Applied Biosystems(Foster City, Calif.). DNA and amino-acid sequence analyses wereperformed with the programs Editbase (Purdue Research Foundation andUSDA/ARS) and Lasergene (DNASTAR Inc., Madison, Wis.).

In verifying the sequences of PCR products, two discrepancies werediscovered near the end of the published cpcB DNA sequence from the sameorganism (5). Base number 487 (numbering of the published sequence) isan A instead of a T. This changes the Cys codon (TGC) to a Ser codon(AGC). Another difference is within the tandem CTG repeats shortlybefore the stop codon. There are four such CTG triplets, rather thanonly three shown in the published sequence. In addition to sequencingPCR-generated clones, the base changes were also confirmed by sequencingof a restriction fragment containing the entire cpcB gene. The last 36base pairs of the cpcB gene sequence determined here, encompassing bothcorrections, encode a 12-amino acid sequence identical to that ofanother filamentous cyanobacterium, Mastigocladus laminosus [(18);GenBank accession No. M75599], and the sequence of the entire CpcBpolypeptide aligns much better with those of other C-phycocyanin βsubunits (19). The mass of the Anabaena sp. PCC7120 β subunit is inexcellent agreement with that predicted from the corrected cpcBsequence. A 16.3-kb DNA sequence with the corrected cpcB sequence, thecomplete sequence of cpcF (see below), and other new information hasbeen deposited into GenBank under accession No. AF178757.

Construction of expression vectors—A family of plasmids was specificallyconstructed for inducible overexpression of His-tagged polypeptides inboth E. coli and Anabaena sp. PCC7120. Plasmid pBS150v contains thefollowing components: (A) From 0 to 1.3 kb, a portion from pBR322 [(20);GenBank accession No. V01119] that contains the ColE1 oriV for plasmidreplication in E. coli, the oriT (bom) site for conjugal transfer, andthe rop (repressor of primer) gene. Although the first 11 codons of theRop open reading frame were changed in pBS150v, the modified Rop proteinapparently can still form the homodimer four-alpha-helix structure (21),helping to maintain the plasmid at a medium copy number like that ofpBR322 (20); (B) From 1.3 to 2.3 kb, most of the C.S4 cassette (22)containing the engineered aadA gene that confers resistance to Sm andSp; (C) From 2.3 to 4 kb, a portion from the expression vector pPROEX-1(Life Technologies, Inc.) that contains the lacI^(q) gene coding for thelac repressor, and multiple cloning sites for construction of sequencescoding for His-tagged proteins, expression of which is controlled by thetrc promoter. The N-terminal 24 residues contain the 6×His affinity tagas well as a 7-amino acid recognition and cleavage site for the tobaccoetch virus (TEV) protease for removal of the 6×His tag if so desired(23, 24); and (D) From 4 to 4.6 kb, a portion from the expression vectorpMAL-c2 (New England Biolabs, Inc.) that contains the lacZ^(α) gene forblue/white screening of insert recombinants, and downstream, two strong,bidirectional, ρ-independent transcriptional terminator structures fromthe E. coli rrnB gene (25). The nucleotide sequence of pBS150v has beendeposited in GenBank under the accession No. AF177932.

A portion of the plasmid pDU1 (26) coding for a replication protein anda resolvase was inserted in the unique Eco47III site of pBS150v, givingplasmid pBS150. This 3.7-kb fragment, pDU1HC, contains spontaneousmutations that enable autonomous replication of cognate plasmids inhigher copy number in Anabaena sp. (27). Plasmid pBS152v (sequence alsoavailable from GenBank under accession No. AF177933) is similar topBS150v but sacrifices the lacZ^(α) gene for more cloning sites likethose in pPROEX-1. The pDU1HC⁺ version, pBS152, was constructed likepBS150. His-tagged constructs were usually made using the small, “v”versions of the plasmids and then converted to the larger,pDU1HC-containing form. When induced, the trc promoter initiates verystrong transcription in both E. coli and Anabaena. While very tightlycontrolled in E. coli, the trc promoter is poorly repressed in Anabaenasp. partially due to the atypical promoter and the presence of somecodons unfavorable to Anabaena sp. in the lacI^(q) gene, leading to arelatively high level of constitutive expression of the 6×His-taggedproteins. Preliminary Western analyses showed only about three-foldincrease in production of His-tagged proteins in Anabaena cultures uponinduction with IPTG.

Isolation of phycobilisomes—Phycobilisome (PBS) preparations werecharacterized on discontinuous sucrose density gradients as described(28), with minor modifications. Frozen or freshly harvested Anabaenacells were used with no obvious difference in results. Na/K-PO₄ buffer(0.75M; 1:1 ratio of Na-PO₄ and K-PO₄, pH 7.5) containing 1 mM each ofNaN₃ and ethylenediamine tetraacetate (EDTA), was used. Individual PBS,rod, and hexamer fractions collected from the sucrose gradients weredialyzed extensively against buffer 0 (20 mM Tris-HCl pH 8.0, 50 mMNaCl, 50 mM KCl) to eliminate sucrose, phosphate, azide, and EDTA beforeisolation of His-tagged phycobiliproteins on a Ni²⁺-NTA column (seebelow). Similarly, for isolation of intact His-tagged PBS on Ni²⁺-NTA,the PBS fraction was dialyzed against 0.75 M phosphate buffer, pH 7.5(without EDTA and azide) prior to loading on a Ni²⁺-NTA column (seebelow).

To isolate PBS containing His-tagged phycocyanin subunits, the PBSpreparation in 0.75 M phosphate was loaded on 1 ml of Ni²⁺-NTA resinpre-equilibrated with 0.75 M phosphate buffer. The high concentration ofphosphate, along with possibly lesser steric availability of the 6×Histags of phycocyanin subunits incorporated within PBS, significantlydecreased the rate of binding of PBS to the Ni²⁺-NTA resin. Severalpassages of the same sample through the column were needed to getadequate binding. The resin was then washed with 20 column volumes of0.75 M phosphate buffer plus 30 mM imidazole. PBS bound on resin wereeluted with 0.75 M phosphate buffer plus 200 mM imidazole. The eluatewas immediately dialyzed against 0.75 M phosphate buffer to removeimidazole.

Isolation of His-tagged Proteins by Immobilized Metal AffinityChromatography—Cell pellets were thawed and resuspended in 10 to 20volumes of cold (0-4° C.) buffer 0. Phenylmethylsulfonyl fluoride (PMSF)and β-mercaptoethanol were added to give final concentrations of 1 mMand 10 mM, respectively, immediately before breakage of cells by passagethrough a French press cell, three times at 18,000 p.s.i. Cellulardebris was then removed by centrifugation at 4° C. in an angled rotor at30,000×g for 20 min (preparations from E. coli) or at 130,000×g for 1 hr(preparations from Anabaena sp.). The supernatant was loaded on a columnof 1 to 3 ml of Ni²⁺-NTA resin pre-equilibrated with buffer 0. The resinwas then washed consecutively with ten column volumes each of coldbuffer A1 (20 mM Tris-HCl pH 8.0, 50 mM NaCl, 50 mM KCl, 20 mMimidazole, 5% v/v glycerol), buffer B (20 mM Tris-HCl pH 8.0, 500 mMNaCl, 500 mM KCl), and buffer A2 (20 mM Tris-HCl pH 8.0, 50 mM NaCl, 50mM KCl, 30 mM imidazole). His-tagged proteins were eluted from the resinwith buffer C (20 mM Tris-HCl pH 8.0, 50 mM NaCl, 50 mM KCl, 200 mMimidazole). The eluate was immediately dialyzed against 50 mM Tris-HClpH 8.0, 50 mM NaCl, 50 mM KCl, 1 mM DTT, to remove imidazole which athigh concentrations is slightly denaturing to proteins. RecombinantHis-tagged phycocyanin isolated in this manner consisted mostly of αβheterodimers. A crude estimate of the yield of His-tagged phycocyaninwas calculated from the amount of phycobiliprotein (expressed in unitsof A_(620 nm) ml) eluted with buffer C vs. the total amount ofphycobiliprotein applied to the Ni²⁺-NTA resin.

Size Exclusion Chromatography-High Performance LiquidChromatography—Although IMAC-purified and dialyzed samples (see above)could be used with no obvious effect on the results, proteins to beanalyzed by SEC-HPLC were generally dialyzed again in 50 mM Na-PO₄ pH7.0, 50 mM NaCl, 0.5 mM DTT. SEC-HPLC separations were performed with aWaters 600 pump and line detection achieved with a Waters 991 photodiodearray detector (Waters Associates, Milford, Mass.). A mobile phase of 50mM Na-PO₄ pH 7.0 was flowed at 0.8 ml min⁻¹ through Bio-Sil 250 (Bio-RadLaboratories, Hercules, Calif.) guard column (80×7.8 mm) and column(300×7.8 mm) maintained at 25° C. For analytical separations, 50 to 200μg of phycocyanin was loaded [based on absorbance at the peak near 621nm and using a molar extinction coefficient of 290,000 M⁻¹ cm⁻¹ permonomer (29)]. Molecular size calibration utilized bovine gammaglobulin, 158 kDa; chicken ovalbumin, 44 kDa; horse myoglobin, 17 kDa;vitamin B12, 1.35 kDa (Bio-Rad gel filtration standards). The voidvolume was determined with blue dextran, 2000 kDa (Pharmacia Biotech,Piscataway, N.J.). Preparative separations were performed with loads of0.5 to 1 mg phycocyanin.

Analytical Ultracentrifugation—Measurements were performed on a BeckmanXLA centrifuge using an An-60Ti rotor maintained at 20° C. and 20,000rpm. Affinity-purified samples were run at a concentration of 0.5 mgml⁻¹ in dialysis buffer. Data were fit to a two component regression(which was superior to a single-component fit) and the molecular weightsdetermined with the HID4000 software.

SDS-Polyacrylamide Gel Electrophoresis—Proteins were precipitated with10% (w/v) trichloroacetic acid, washed once with ice-cold water, andresolubilized in SDS loading buffer [2% sodium dodecyl sulfate, 50 mMTris-HCl, pH 6.8, 100 mM DTT, 0.1% Bromphenol Blue, and 10% v/vglycerol; (13)]. SDS-PAGE (30) was performed using 10% acrylamidestacking and 14% separating gels, with monomer/bis ratio of 37.5:1.Bilin-bearing polypeptides were visualized as fluorescent bands by UV(>312 nm) illumination after staining with 10 mM zinc acetate (31).Polypeptides were also visualized by staining with Coomassie BrilliantBlue. Protein molecular weight standards were purchased from Bio-RadLaboratories.

Absorbance and Fluorescence Spectrometry—Absorbance spectra wereacquired on a computer-controlled, dual-beam λ6 UV/V isspectrophotometer (Perkin-Elmer Corp., Norwalk, Conn.). Absorbance ofAnabaena cell suspensions was measured from 400 to 750 nm with a lightbeam passing through the frosted side of a 1-cm light path squarecuvette. Fluorescence measurements were performed, on samples with amaximal absorbance of less than 0.07 to minimize the inner-filtereffect, with a Perkin-Elmer MPF-44B fluorimeter coupled with an I/Oboard to a Macintosh computer for digitization and storage of data.Excitation and emission slits were set at 5 or 6 nm for allmeasurements. Excitation spectra were measured with emission observed ata wavelength 10 nm to the red of the sample's fluorescence emissionmaximum.

Fluorescence quantum yield measurements, for samples isolated fromSEC-HPLC, were made relative to cresyl violet (in ethanol, Φ_(f)=0.59;Eastman Kodak Co., Rochester, N.Y.) using the K2 multifrequency phaseand modulation fluorimeter (ISS, Champaign, Ill.) with excitation andemission slits set at 8 nm. All samples were diluted to peak absorbancebetween 0.01 and 0.05, excited at 570 nm and emission acquired from 575to 800 nm. These emission spectra were then instrument-corrected,converted to wavenumber scale, and bandpass corrected by multiplyingemission intensity at each wavelength by the square of the respectivewavelength (32). The resulting spectra were integrated and quantumyields calculated according to previously described equations (33).

Molar extinction coefficients were determined according to previouslyestablished methods (34). In brief, absorbance spectra of samplesisolated as trimers (unless otherwise noted) from SEC-HPLC weremeasured. These samples, having peak absorbance between 0.9 and 1.2,were diluted 10× with 8 M urea, pH 1.9 containing 10 mM DTT. Theabsorbance was measured at 660 nm for these denatured samples, and molarextinction coefficients were calculated for the original native samplesfrom the known value of 35,400 M⁻¹ cm⁻¹ (at 660 nm) for eachpeptide-bound PCB in acid urea (35). Calculations assumed astoichiometry of one PCB per α subunit and two PCBs per β subunit. Molarextinction coefficient of each peptide-bound PCB at 280 nm was measuredusing trimeric phycocyanins purified from SEC-10 HPLC. Samples in 50 mMNa-PO₄ pH 7.0, with peak absorbance between 1.0 and 1.5 at 620 nm, weredenatured with 9 M urea to give final 8 M urea, pH 2.0. The extinctioncoefficient of a protein-bound PCB at 660 nm was identical to the valuementioned above, while that at 280 nm was calculated by subtractingcontributions from Tyr [ε_(280 nm)=1,370 M⁻¹ cm⁻¹; (36)] and Trp[ε_(280 nm)=5,500 M⁻¹ cm⁻¹; (37)] residues.

Mass Spectrometry—For electrospray mass spectrometry, purified proteins(0.5 to 1 mg) were dialyzed extensively against 10 mM ammonium acetateprior to lyophilization. Proteins redissolved in 50% acetonitrile in0.2% aqueous formic acid were analyzed with a VG Bio-Q mass spectrometeras described (38). Mass spectral analysis of different phycobiliproteinpolypeptides, as well as of some other proteins expressed in Anabaenasp., showed quantitative posttranslational removal of the N-terminal Metin Anabaena sp. PCC7120. For this reason, amino acid residue numberingstarts from the residue after the initial Met, and follows that used fornumbering of bilin-linked residues in crystallographic studies of thehighly homologous phycocyanin from Mastigocladus laminosus (39).However, our examination of phycobiliproteins from other cyanobacteria,as well as amino acid sequence data published by others (40, 41), showedthat such posttranslational processing is not universal tocyanobacteria.

Molecular modeling of His-tagged C-phycocyanin—The crystal structure ofC-phycocyanin from the cyanobacterium Fremyella diplosiphon has beensolved at 1.75 Å resolution (42). The structural data for thatphycocyanin, which is highly homologous to that of Anabaena sp. PCC7120,were used to build a molecular model of the Anabaena C-phycocyanin withthe 24-amino acid N-terminal extension on the α subunit. Examination ofthe C-phycocyanin hexamer structure revealed a groove lying on the βface between β subunits. In order to present a reasonable graphicalrepresentation of the HTα:β model, we chose to lead the 24-amino acidextension from inside to outside of the ring structure of phycocynintrimer through this surface groove. Although structural data supportingthis choice is lacking, our results suggest that this is a plausibleplacement. Modeling of the 24-residue extension onto the C-phycocyaninhexamer was performed using the published monomer coordinates [(42); PDBaccession code: 1cpc]. The 24-residue tag was built onto the N-terminusof the α subunit as follows. First, the coordinates for a well-defined25-residue peptide (the last one at C-terminus being Met) were excisedfrom the published coordinates of another protein (antibody 48G7; PDBaccession code: 1GAF). Next, all side chains except for that of the lastMet were truncated to Ala (or left as Gly). Using the least-squaressuperposition of the program LSQMAN (43), the C-terminal Met of the25-mer peptide was overlaid with the N-terminal Met of the at subunit .The tag was then coarsely laid into the above-mentioned groove using thetorsion commands of the program O (44), and the side chains mutatedagain to conform with sequence of the 24-residue tag of HTα. Again,using only the torsion commands, phi, psi, and chi angles on the24-residue tag were manipulated so as to conform to the followingconstraints: (a) all non-bonded contacts involving tag-to-tag andtag-to-phycocyanin atoms must be in excess of 2.5 Å; (b) large part ofthe tag must lay more or less in the β-face groove; and (c) the six Hisresidues must place externally to the hexamer so as to besolvent-accessible. The model was then subjected to 200 rounds of Powellenergy minimization using the program XPLOR (45, 46), during which allof the original C-phycocyanin atoms were constrained from movement. Thisresulted in the torsion angles and non-bonded contacts of the 24-residuetag approaching canonical values. Quality of the model with respect tophi/psi angles was verified using the program PROCHECK (47). TheRamachandran plot showed that the modeled tag contained no disallowedphi or psi angles. To build trimers, symmetry operators were applied tothe HTα:β monomer model using program O. The hexamer model was built byadding the tag coordinates to the second α subunit coordinates of the1cpc structure and then applying the symmetry operators.

Nomenclature—A His-tagged protein preparation purified by IMAC maycontain more than one protein species. For example, both the apoproteinand holoprotein version of a His-tagged phycocyanin subunit may bepresent. Consequently, we refer to such a preparation by a nomenclaturewhich specifies the organism in which it is being expressed (i.e.,Anabaena sp. or E. coli), and the number of the pBS plasmid whichcontains the gene encoding the recombinant subunit. Thus, whenHis-tagged phycocyanin α subunit is expressed in Anabaena sp. PCC7120bearing the plasmid pBS168, the fraction purified by IMAC is designatedAn168. A fully characterized fraction, shown to consist of a singlespecies, for example, pure His-tagged phycocyanin α subunit, isdesignated HTα. A mutant polypeptide, for example, a His-tagged Anabaenasp. PCC7120 phycocyanin α subunit with a Thr residue in place of an Alaresidue at position 12, is designated HTα^(A12T). The absence orpresence of the bilin chromophores is indicated by the prefixes “apo”and “holo”, respectively. A His-tagged α subunit with native β subunitnon-covalently associated is designated HTα:β.

Table 1 summarizes the properties of each of the Anabaena sp. PCC7120wild-type and derivative strains and of expression vectors carried bythese strains used for the production of His-tagged C-phycocyanin normaland mutant α and β subunits.

Expression of His-Tagged C-Phycocyanin α Subunit in Wild-Type Anabaenasp.—The 0.5-kb NdeI-cpcA-HindIII fragment from pBS185, encoding thewild-type phycocyanin α subunit, was cloned into pBS150, giving plasmidpBS168 encoding the α subunit with a 24-residue N-terminal extension.Cultures of Anabaena sp. PCC7120(pBS168) expressing 6×His-taggedphycocyanin α subunit were very similar to wild-type cultures grownunder identical conditions with respect to the relative amounts ofphycobiliproteins and chlorophyll, and showed no apparent negativephenotypic characteristics.

Isolation and characterization of HTα—When cell lysate supernatant fromAnabaena sp. PCC7120(pBS168) was passed through a Ni²⁺-NTA affinitycolumn, some 30 to 40% of the cell phycobiliprotein (as estimated fromA_(max) at 620 nm) was retained on the column and then eluted withimidazole, a His-tag competitor. SDS-PAGE analysis of the His-taggedprotein fraction showed two bands of 21.9 kDa and 19.5 kDa,corresponding to the calculated molecular weights of His-taggedC-phycocyanin α and native β subunits, respectively. Upon Zn²⁺-staining,both bands showed red fluorescence with near-UV excitation, indicatingthat each carried covalently linked phycocyanobilin. Identification ofthe two polypeptides was confirmed by mass spectrometry, which showedtwo components of 20,791.4±2.3 (holo-HTα minus the N-terminal Met) and19,441±1.3 (holo-β minus the N-terminal Met) in a 1:1 ratio. These massvalues confirm the presence of one PCB on HTα and two PCBs on the βsubunit. No other significant peaks, especially that corresponding to anapo-HTα, were present in the mass spectrum. These data indicate that theprotein purified by IMAC, An168, is phycocyanin holo-HTα:holo-β.

The assembly forms of phycocyanin holo-HTα:holo-β and their apparentmolecular weights were determined by analytical SEC-HPLC. As shown inTable 2, three components were present under our particularchromatographic conditions, a monomer (calculated mass 40.5 kDa) at 49.2kDa, a trimer (calculated mass 121.5 kDa) at 145.3 kDa, and a hexamer(calculated mass 243 kDa). Over 90% of the protein was trimeric,(HTα:β)₃, the major assembly state observed under these conditions fornative PC isolated from wild-type Anabaena sp. PCC7120. The SEC-HPLCdata were corroborated by analytical ultracentrifugation, where most ofthe HTα:β was found to have a mass of 125.9 kDa, and the balance a massof 34.8 kDa (Table 2). All three fractions from SEC-HPLC were shown bySDS-PAGE to contain holo-HTα and holo-β in a ratio of 1:1. Thus, theAn168 His-tagged protein preparation purified by IMAC consists of HTα:β,and displays aggregation behavior typical of native PC under similar invitro conditions.

(HTα:β)₃ isolated by SEC-HPLC had a λ_(max) of 621 nm at proteinconcentrations >0.5 mg ml⁻¹, and gave spectra with blue-shifted maxima(to as low as 615 nm) upon dilution (Table 3). This is consistent withthe blue shift in λ_(max) of wild-type phycocyanin when higher orderassemblies dissociate to monomers as the protein concentration islowered (48). Interestingly, at high protein concentrations, HTα:βdisplays a λ_(max) slightly red-shifted relative to that of native PCmeasured at similar concentrations. The ε_(M) of (HTα:β)₃ was determinedto be 9.21×10⁵ M⁻¹ cm⁻¹ at 621 nm, similar to values observed for nativephycocyanin from Anabaena sp. and other cyanobacteria [Table 3; (49)].The excitation spectrum for 650 nm emission coincided nearly perfectlywith the absorbance spectrum, indicating that the HTα:β preparationbehaved as a single species with respect to spectroscopic properties.The λ was at 642 nm (with 560 nm excitation) with a Φ_(F) of 0.22,similar to a value of 0.27 measured for native Anabaena PC (Table 3). Insummary, the spectroscopic characteristics of HTα:β are virtuallyidentical to those of native Anabaena sp. PCC7120 C-phycocyanin.

Incorporation of HTα into Phycobilisomes—Since holo-HTα:holo-βrepresents between 30 and 40% of the total phycobiliprotein in Anabaenasp. PCC7120(pBS168), it appeared likely that holo-HTα is effectivelyincorporated into phycobilisomes. This possibility was examined byanalyzing phycobilisomes and their partial dissociation products for thepresence of holo-HTα. Compared to the wild-type Anabaena sp. PCC7120phycobilisome preparation, in the Anabaena sp. PCC7120(pBS168)preparation a lower percentage of the total phycobiliproteins was foundin the phycobilisome fraction, while more was found in the rodsubstructure and the hexamer fractions. This indicates that thephycobilisomes of the strain expressing holo-HTα are less stable underthe conditions used for phycobilisome preparation. SDS-PAGE analysisshowed that holo-HTα was present in all three fractions.

Anabaena sp. PCC7120(pBS168) phycobilisomes loaded on a Ni²⁺-NTA column(in the 0.75M Na/K-PO₄ pH 7.5 buffer needed to maintain phycobilisomeintegrity, see “Materials and Methods”), bound to the column, althoughthe binding capacity of the column under these conditions appeared low.The His-tagged phycobilisomes were eluted at high imidazoleconcentration. After removal of imidazole by dialysis, the SDS-PAGEpolypeptide profile and spectroscopic properties of this phycobilisomefraction were very similar to that of the starting phycobilisomepreparation. These results show that the 6×His tags of the HTα subunitsincorporated into phycobilisomes are relatively exposed, available forinteraction with the Ni²⁺-NTA matrix.

Expression of His-Tagged C-Phycocyanin β Subunit in Wild-Type Anabaenasp.—The 0.5-kb NdeI-cpcB-EcoRI fragment from pBS251, encoding thewild-type phycocyanin β subunit, was cloned into pBS150, giving plasmidpBS262 encoding the β subunit with the 24-residue N-terminal extension.Cultures of Anabaena sp. PCC7120(pBS262) expressing HTβ werephenotypically very similar to those of Anabaena sp. PCC7120(pBS168)expressing HTα, except for a slightly yellowish appearance. Theirwhole-cell absorbance spectra showed a small decrease in the amount ofphycobiliprotein per cell relative to chlorophyll.

The yield of the His-tagged phycobiliprotein fraction from Anabaena sp.PCC7120(pBS262) was 16-18% of the total phycobiliprotein in the celllysate, substantially lower than the 30-40% in the correspondingfraction from Anabaena sp. PCC7120(pBS168) expressing HTα (see above).Analysis of the His-tagged phycobiliprotein preparation, An262, bySDS-PAGE showed the presence of holo-HTβ and holo-α in equal amounts.Mass spectral analysis showed that the two subunits carried the normalamounts of PCB, i.e., one PCB per α and two per HTβ subunit, and noapo-polypeptides were present.

In analytical SEC-HPLC, the α:HTβ holoprotein preparation isolated byIMAC was primarily trimeric (calculated mass 121.5 kDa) at 136.9 kDa.The shoulder/tail of that peak, constituting less than 5% of the totalHis-tagged protein loaded, could be attributed to monomers (calculatedmass 40.5 kDa) at 62.4 kDa (Table 2). Both fractions were shown bySDS-PAGE to contain holo-α and holo-HTβ in a molar ratio of 1:1. Incontrast to native PC and the HTα:β holoprotein, the hexamer componentwas not observed for the α:HTβ holoprotein. Analyticalultracentrifugation data were consistent with two components observed inSEC-HPLC: a majority of the α:HTβ holoprotein had a mass of 121.9 kDa,and a small fraction of 43.4 kDa, corresponding closely to thetheoretical masses of trimers and monomers, respectively.

The λ_(max) of the trimeric α:HTβ holoprotein component lay between 615and 619 nm depending on protein concentration (Table 3). As observedwith HTα:β, λ_(max) shifted blue upon dilution. However, at high proteinconcentration the λ_(max) of the α:HTβ holoprotein, like that of nativePC, did not exceed 619 nm, in contrast to the λ_(max) of 621 nm observedfor HTα:β under similar conditions. The ε_(M) of α:HTβ trimers was foundto be 8.88×10⁵ M⁻¹cm⁻¹ at 619 nm (Table 2), a slightly lower value thanthat observed for HTα:β. The excitation spectrum for 650 nm emissioncorresponded nearly perfectly to the absorbance spectrum, indicatingthat the α:HTβ preparation behaved as a single species with respect tospectroscopic properties. The λ was at 642 nm (with 560 nm excitation),with a Φ_(F) of 0.23 (Table 2). Thus the spectroscopic characteristicsof α:HTβ are virtually identical to those of native Anabaena sp. PCC7120C-phycocyanin and its HTα:β counterpart.

A sucrose density gradient preparation of phycobilisomes from Anabaenasp. PCC7120(pBS262) expressing HTβ showed distribution ofphycobiliprotein aggregates different from that of the wild-type cells.A much smaller proportion of the phycobiliprotein (25%) sedimented inthe phycobilisome fraction, with 40% and 35% in the rod and hexamerfractions, respectively. SDS-PAGE analysis showed that HTβ was presentin all three fractions. However, less HTβ (relative to the otherphycobiliproteins) was found in phycobilisomes compared to the amountfound in the products of phycobilisome dissociation, rods and hexamers,suggesting a reduced stability of phycobilisomes that have incorporatedmore HTβ subunits. As described above for Anabaena sp. PCC7120(pBS168)expressing HTα, His-tagged phycobilisomes could be isolated from theAnabaena sp. PCC7120(pBS262) phycobilisome fraction by IMAC, although ina relatively poor yield. The low yield might be due to the smallernumber of HTβ subunits present per phycobilisome, a reflection of thesmaller amount of holo-HTβ relative to holo-β produced per cell.

Highly purified (HTα:β)₃ and (α:HTβ)₃ phycocyanins were used to measurethe extinction coefficient of peptide-bound PCBs in 8 M urea pH 2.0. Twodifferent preparations were measured for each protein, and fourmeasurements gave very similar values. The molar extinction coefficientfor one peptide-bound PCB at 660 nm is very close to the value of 35,400M¹ cm⁻¹ (35). Upon subtraction of contributions from Trp and Tyrresidues, the extinction coefficient for one peptide-bound PCB at 280was determined to be (14.85±0.1)×10³ M⁻¹ cm⁻¹ (Table 3).

Expression of HTα and HTβ in a cpcBAC background—The separateoverexpression of HTα and HTβ in phycocyanin-minus backgrounds wasexamined in mutant strain B646 (7). This mutant has a transposoninserted between the promoter and the CpcB open reading frame, which isexpected to eliminate transcription of cpcBAC, genes encoding the β andα subunits of phycocyanin and a rod linker polypeptide, respectively(5). Strains B646(pBS168) and B646(pBS262) (see Table 1), generated byintroduction of pBS168 and pBS262 into strain B646 by conjugation, werephenotypically indistinguishable from the parent mutant strain. Bothphycocyanin-deficient strains grow slowly and produce very low levels ofthe His-tagged polypeptides.

SDS-PAGE analysis of the His-tagged phycobiliprotein fractions fromstrain B646(pBS262) showed no α or β subunits of wild-type phycocyanin.However, multiple attempts at purifying the HTα from strain B646(pBS168)yielded fractions containing some native β subunit, presumably resultingfrom a basal leaky transcription of the cpcBAC operon.

On SEC-HPLC fractionation, the IMAC-purified proteins from B646(pBS262),An262-BAC, eluted as a single peak of subunit homodimers, i.e., (HTβ)₂.Purified native β subunits of C-phycocyanin dimerize at concentrationsof 1-10 μM (34, 50). Since mass spectrometry showed that the HTβsubunits produced in this cpcBAC background carried a full complement ofPCB, it is evident that bilin addition to phycocyanin β subunit isindependent of the presence of the phycocyanin apo- or holo-α subunit.The mass spectral analysis also indicates the presence in HTβ of theposttranslational methylation at the γ-amino group of the Asn⁷² residuenormally found in phycocyanin β subunits (51, 52).

Under similar SEC-HPLC conditions IMAC-purified protein preparation fromB646(pBS168), An168-BAC, fractionated into three species: trimer,monomer, and subunit. SDS-PAGE analysis of the fractions indicated thatthe trimer and monomer fractions contained HTα and holo-β in equimolaramounts. The subunit fraction contained only HTα, the majority of whichappeared to be apo-HTα based on the relative intensities in theabsorption peaks at 280 nm and 618 nm, and from comparison of Zn²⁺—andCoomassie-staining intensities of the An168-BAC trimer and monomerfractions (HTα:β) run in parallel on the same gel.

Analyses of the His-tagged protein preparations from strainsB646(pBS168) and B646(pBS262) indicate that overexpression of HTβ in thecpcBAC background yields a soluble protein where the HTβ subunit isstabilized by homodimer formation. The results also indicate that HTαappears to be unable to form subunit homodimers in the cell, and mayrequire the presence of β subunit that is extremely limited in thecpcBAC background for stabilization. The greatly reducedchromophorylation of HTα protein isolated from this cpcBAC strain mayalso reflect a possible requirement of subunit dimerization (α:β, α:α,or β:β) prior to covalent attachment of PCB, or misfolding of asignificant fraction of apo-HTα in the absence of apo- or holo-β.

The HTα and HTβ subunits isolated from mutant strains B646(pBS168) andB646(pBS262) have absorbance spectra and molar extinction coefficients(Table 3) similar to those reported for renatured α and β subunits ofnative C-phycocyanin, obtained by chromatographic separation underdenaturing conditions (34, 50). Algebraic addition of the HTα and HTβspectra yielded a spectrum with shape similar to that of monomeric PCbut with a blue-shifted maximum at 611 nm, consistent with previousobservations (34). Incubation of the HTα and HTβ subunits togetherovernight at 40° C., followed by fractionation by SEC-HPLC, yielded atrimer fraction with absorbance and fluorescence excitation and emissionspectra similar to the corresponding spectra of HTα:β, α:HTβ, and nativePC. This indicates that holo-HTα (but not apo-HTα, see below) interactspreferentially with holo-HTβ to form trimers, and His-taggedholophycocyanin subunits isolated in the absence of their cognatesubunit partners can be reconstituted to yield trimers with propertiessimilar to those of native phycocyanin.

Expression of His-tagged phycocyanin subunits in an apophycocyanin αsubunit PCB lyase-deficient background—A dimeric lyase, CpcEF encoded bygenes cpcE and cpcF, specifically catalyzes the addition of PCB to Cys⁸⁴of the phycocyanin α subunit (1, 3). We have obtained two mutantsthrough transposon mutagenesis, B64328 with an insertion in the cpcEgene, and B64407 in the cpcF gene. Both mutants had greatly reducedcellular content of normal phycocyanin (7). Expression of His-taggedphycocyanin subunits in the mutant strains provided a simple way ofassessing how mutations in each of the two CpcEF lyase subunits affectPCB addition to the phycocyanin α subunit, and how cognate PC proteinsbehave in the cell.

Expression of HTα in either a cpcE or a cpcF background—To express theHTα polypeptide in either a cpcE or cpcF background, plasmid pBS168 wasintroduced into both mutants, giving strains B64328(pBS168) andB64407(pBS168) (see Table 1). The two strains showed the yellowish-greenphenotype characteristic of their respective parent mutants. TheHis-tagged phycobiliprotein fraction from either mutant strain amountedto <5% of the total absorbance of the cell lysate at 607 nm. TheHis-tagged protein purified from the cpcF strain B64407(pBS168),An168-F, was shown by SDS-PAGE and mass spectral analyses to beapo-HTα:holo-β, with no detectable holo-HTα. In contrast, the An168-Eprotein contained apo-HTα:holo-β along with a small amount ofholo-HTα:holoβ. On SEC-HPLC, apo-HTα:holo-β is seen to form monomerspreferentially, in contrast to the preferred trimer formation byholo-HTα:holo-β.

Sucrose density gradient sedimentation patterns of phycobilisomepreparations from strains B64328(pBS168) and B64407(pBS168) were verysimilar to those given by the parental strains B64328 and B64407 (7). Inboth cases, the phycobilisome fraction consisted largely of theallophycocyanin-containing phycobilisome cores carrying much reducedamounts of rod components (relative to phycobilisomes from wild-typecells). SDS-PAGE analysis showed the presence of apo-HTα in thephycobilisome and rod/hexamer fractions and an amount ofphycoerythrocyanin, a distal component of Anabaena phycobilisome rodsubstructures, greater than that seen in wild-type phycobilisomes (7,53). The relative amount of apo-HTα was higher in the phycobilisomefraction than in the rod/hexamer fraction, suggesting that apo-HTαassembled into phycobilisomes might be turned over more slowly in thecell.

The finding that apo-HTα:holo-β assembles into phycobilisomes isconsistent with earlier observations of such incorporation of thephycocyanin apo-α subunit in cpcE and cpcF mutants of the unicellularcyanobacterium Synechococcus sp. PCC7002 (3, 4). We showed previouslythat the Anabaena sp. PCC7120 mutant strains B64328 (cpcE) and B64407(cpcF) incorporated the phycocyanin apo-α:holo-β into phycobilisomes,and that this “defective” phycocyanin still allowed the assembly intophycobilisomes of the distal phycoerythrocyanin component (7). Theexperiments reported here extend that observation to phycocyaninapo-HTα:holo-β proteins.

Expression of HTβ in either a cpcE or a cpcF background—To express theHTβ polypeptide in either a cpcE or cpcF background, plasmid pBS262 wasintroduced into both mutants, giving strains B64328(pBS262) andB64407(pBS262) (see Table 1). Yield of His-tagged holophycobiliproteinsfrom either mutant strain was low, accounting for <7% of the absorbanceof the cell lysate at 607 nm. In contrast to results obtained with themutant strains expressing HTα (see above), when HTβ was expressed,SDS-PAGE analysis showed that the His-tagged protein preparationscontained apo-α and holo-HTβ in a molar ratio of approximately 1:2.SEC-HPLC fractionation showed presence of apo-α:holo-HTβ trimers andmonomers, and of (holo-HTβ)₂ homodimers. The apo-α:holo-HTβ trimers hadspectroscopic properties virtually identical to those of apo-HTα:holo-βtrimers described above. Analyses of phycobilisomes and of rod/hexamerfractions showed that some of the holo-HTβ (likely in the form ofapo-α:holo-HTβ) was incorporated into both fractions.

Expression of His-tagged mutant phycocyanin subunits—The favorableexpression and incorporation of His-tagged wild-type phycocyaninsubunits in Anabaena sp. provides a convenient means to perform in vivoand in vitro studies of mutant phycobiliprotein subunits. Here wepresent two examples.

Expression of HTα^(A12T)—One of the cpcA clones generated by PCR usingthe Taq DNA polymerase was found to have a single G→A base changeleading to an Ala¹²→Thr mutation in the translated protein. The Ala¹²residue is conserved in all sequenced phycocyanin α subunits (19). Thecrystal structure of phycocyanin predicts that replacement of Ala¹² by aresidue with a larger side chain would interfere with αβ heterodimerformation by steric hindrance of the interaction of α-Asp¹³ with β-Tyr⁹⁷(54). We expressed the HTα^(A12T) mutant subunit in wild-type Anabaenasp. PCC7120 to examine this prediction.

The mutant cpcA gene was cloned into pBS1350, giving plasmid pBS1167.Anabaena sp. PCC7120(pBS167) expressing HTα^(A12T) was phenotypicallyvery similar to strain Anabaena sp. PCC7120(pBS168) (see Table 1).However, the His-tagged protein fraction was obtained in very poor yieldfrom Anabaena sp. PCC7120(pBS167): <0.15% of the phycobiliproteins inthe cell lysate as estimated from A_(620 nm). While spectroscopicallysimilar to the An168 His-tagged protein preparation, mass spectralanalysis of the An167 His-tagged protein preparation showed twocomponents in similar amounts, with masses corresponding to those ofholo-α and holo-HTα^(A12T). These results lead to two conclusions.First, the mutant HTα^(A12T) subunit appears to have a greatly reducedaffinity for the holo-β subunit. Most of the HTα^(A12T) is likely to bepresent as free subunits in the cell and to be degraded rapidly.HTα^(A12T) also seems to have a low affinity for holo-α, and isconsequently stabilized in vivo by the formation ofholo-α:holo-HTβ^(A12T) dimers. Second, the in vivo covalent attachmentof PCB to the phycocyanin α subunit evidently does not require that itform a complex with the β subunit.

Sucrose density gradient preparation of phycobilisomes from strainAnabaena sp. PCC7120(pBS167) gave a banding pattern similar to that fromthe wild type. The HTα^(A12T) subunit was not detected in phycobilisomeand rods fractions, suggesting that HTα^(A12T) was not assembled intothe phycobilisomes (although the very low amount of HTα^(A12T) may haveevaded detection).

Expression of HTβ^(S46G,N76D)—One of the cpcB fragments, PCR-amplifiedusing the Taq DNA polymerase, contained two mutations: both an A→Gchange, in bases number 139 and 229, respectively, of the publishedsequence (5). The changes resulted in the replacement of residue Ser⁴⁶with Gly, and of residue Asn⁷⁶ with Asp in the CpcB protein. Anabaenasp. PCC7120(pBS162) expressing the HTβ^(S46G,N76D) mutant PC subunit wasphenotypically identical to the one expressing His-tagged wild-type βsubunit. The HTβ^(S46G,N76D) mutant protein behaved the same way as theHis-tagged wild-type counterpart in all aspects tested (yield, assemblystates in vitro, assembly into phycobilisome in vivo, etc.) except thatthe purified protein, An162, had an absorption maximum at 610 nm, about8-nm blue-shifted compared to that of the An262 protein. Fluorescenceemission maximum of the An162 protein, however, was still at 642 nm.

Ser⁶ of the phycocyanin β subunit, situated in the coil regionconnecting helices A and B (39, 54), is not in contact with anychromophore. Its non-conserved substitutions include Gly in somephycobiliprotein subunits. It therefore may be assumed that theSer⁴⁶→Gly change would not have obvious effects on the protein. TheAsn⁷⁶ residue, on the other hand, is highly conserved amongmulti-chromophore β subunits of phycobiliproteins (19), and itssidechain is in contact with ring D of the α-84 PCB chromophore of PCtrimers, possibly involved in maintaining its stretched confirmation intrimer aggregates (55). The neighboring Arg⁷⁷ residue, also highlyconserved, forms a hydrogen bond with the propionic acid substituent ofring B of the β-84 PCB (54, 55). In the mutant protein, replacement ofAsn⁷⁶ by Asp⁷⁶ sidechain may interfere with that hydrogen bonding,thereby affecting absorbance of β-84 PCB. A reduction of absorbance ofβ-84 PCB would shift the λ_(max) of the cognate phycocyanin to the blue(50).

Molecular modeling of His-tagged C-phycocyanin—Based on crystallographicdata obtained from the cyanobacterium Fremyella diplosiphonC-phycocyanin (42) that is highly homologous to that of Anabaena sp.PCC7120, a molecular model was built for the Anabaena sp. PCC7120C-phycocyanin incorporating the 24-residue N-terminal extension. In thisparticular model, the N-terminal extension can be led from inside tooutside of the ring structure of the phycocyanin trimer, through agroove between β subunits on the β side of the trimer. This allowsunhindered α face-to-α face stacking of two trimers to form a hexamer.The 24-residue N-terminal tag, occupying only the groove space in the βfaces, also does not appear to interfere with stacking of hexamers on βfaces to form rods of the phycobilisome. The first N-terminal 13 of the24 residues of the tag, that include the six His residues, arecompletely exposed outside of the rod surface, consistent with theobserved affinity purification of phycobilisomes by IMAC (as describedabove).

In summary, methodology developed in this study allows (a) high levelexpression of holo-HTα and holo-HTβ in Anabaena sp. PCC7120 and avariety of its mutant derivatives; (b) facile affinity purification ofthe His-tagged polypeptides and of complexes into which they areincorporated, including intact phycobilisomes; (c) study of in vivoassembly and bilin addition; and (d) analysis of site-specific mutantsof C-phycocyanin. It is evident that the approaches described here areimmediately generalizable to other Anabaena phycobiliproteins, such asallophycocyanin and phycoerythrocyanin (3, 7, 29).

With appropriate choice of organism, these approaches can be applied tothe expression and study of other phycobiliproteins includingphycoerythrins. Finally, the studies described here provide for thedesign and expression of diverse fusion proteins in Anabaena sp. withimportant outcomes for the successful production of modular fluorescentphycobiliprotein tags and for the exploration of heterologous proteinfolding in cyanobacteria (9, 10).

Example B. Recombinant Phycobiliprotein Fusion Proteins WithOligomerization and Biospecific Recognition Domains

Here we describe the design and expression of more complex recombinantphycobiliprotein constructs which incorporate oligomerization andbiospecific recognition domains and in some of which the α and βsubunits are covalently bridged. Materials and methods essentially asdescribed in Example A are not restated.

Assays of protein binding to streptavidin—Western hybridization wascarried out essentially as described (14). Proteins separated onSDS-PAGE were transferred to polyvinylidene difluoride (PVDF) membrane(Immobilon; Millipore Corp., Bedford, Mass.) using a mini Trans-Blotcell (BioRad Laboratories). Electrophoretic blotting was carried out inice-cooled transfer buffer for 1 hr at 100 V. The PVDF membrane was thenblocked with 3% bovine serum albumin in PBS buffer (115 mM NaCl, 4 mMKH₂PO₄, 16 mM Na₂HPO₄, pH 7.4) plus 0.5% Tween-20 (Sigma Chemical Co.).Binding of membrane-bound proteins to streptavidin was assayed byincubating the membrane at 25° C. for 1 hr in PBS buffer plus 0.1%Tween-20 and 1:5000-diluted streptavidin-alkaline phosphatase conjugate(Prozyme Inc., San Leandro, Calif.). Alkaline phosphatase-mediated colordevelopment on the membrane using chromogenic substrates5-bromo-4-chloro-3-indolyl phosphate (BCIP) and nitro blue tetrazolium(NBT) was carried out as described (14) except that 200 mM Tris-HCl pH8.8 was used as the developing buffer.

Alternatively, streptavidin-coated agarose beads were used to testbinding of Strep2-tagged phycocyanin. As has been described (56), beadscoated with recombinant core streptavidin (Biometra Inc., Tampa, Fla.)gave better results than those coated with natural streptavidin (SigmaChemical Co. or Prozyme Inc.). Streptavidin-coated beads were incubatedat 4° C. for about 20 min with excess amount of purified Strep2-taggedphycocyanin, washed twice with buffer W (100 mM Tris-HCl pH 8.0, 1 mMEDTA), and visualized by fluorescence. Phycocyanin-labeled beads wereobserved either in batch with ultraviolet (>312 nm) excitation offluorescence viewed through a 550 nm long-pass filter, or under anepifluorescence microscope (the BH-2 system from Olympus America Inc.,Lake Success, N.Y.) with excitation light through a 450-480 nm band-passfilter and emission observed through a 515 nm long-pass filter.

Construction of modular cloning vectors for protein expression—A seriesof cloning and expression vectors was designed and constructed forexpression of fusion proteins with different functional domains and tagson the N-terminus. To facilitate shuffling of the functional domains toproduce desired combinations, DNA cassettes coding for these domainswere designed as exchangeable modules. Care was taken in the design toensure that only optimal codons (in respect to E. coli and Anabaena sp.PCC7120) were used, and that no restriction sites (for 6-bp cutters)were present in the core sequences, except for designed-in signaturesites.

The expression vector pBS150v [Example A; GenBank Accession numberAF177932] was used as a template in the engineering of functionaldomains. Generally, a pair of designed oligonucleotide primers was usedto run inverse PCR, producing a new plasmid in which the sequence frombp 3892 to 3938 (encoding the 6×His affinity tag and a spacer) ofpBS150v is replaced by a desired functional module.

Replacement with the 56-bp NcoI-BspMII Strep2 module generated plasmidpBS283v (4,644 bp; Table 4). The 10-residue Strep2 tag is able to bindspecifically to streptavidin (57).

Replacement with the 146-bp NcoI-AgeI combination module (6×His plusGCN4-pII) gave plasmid pBS311v (4,734 bp; Table 4), and with the 164-bpcombination module (Strep2 plus GCN4-pLI) gave pBS303v (4,752 bp; Table4). Sequences for the GCN4-pII and GCN4-pLI coiled coil domains weremodified from part of the original Saccharomyces cerevisiae GCN4sequence encoding the leucine zipper domain (58, 59), to conform withthe pII sequence (positions a and d are both Ile) or the pLI sequence(positions a and d are Leu and Ile, respectively) (60). A BglII site wasengineered into the GCN4-pII coding sequence as a signature site, as wasan AsuII site in GCN4-pLI.

With plasmids pBS283, pBS303, and pBS311 in hand, four functionaldomains, 6×His, Strep2, GCN4-pII, and GCN4-pLI, are available forfurther manipulation. Since the different functional domains are modularin design, different cloning and expression plasmids with variouscombinations of the modules were easily made by simple recombinationcloning (Table 4). All resulting plasmids have the modular region endwith the second half of either the BspMII or the AgeI site, encoding aGly, immediately upstream from the recognition and cleavage sequence forthe tobacco etch virus (TEV) endoprotease (Example A, herein, 23, 61).The 7-residue TEV site was retained in all plasmids to act as a spacerbetween the modular functional domains and the multiple cloning sitesinto which genes of interest are inserted, and to provide a means ofremoving the modular functional domains from expressed fusion proteinsif so desired. The LacZ^(α) domain downstream from the TEV site wasfound to be functional in all cognate plasmids, allowing blue/whitescreening of inserts in the multicloning region. The multicloning regionof these plasmids can be greatly enhanced in lieu of the LacZ^(α)function by recombination with plasmid pBS152v [(Example A, herein);GenBank Accession number AF177933]. For plasmids encoding phycocyaninsubunits, the NdeI-cpcA-HindIII and NdeI-cpcB-EcoRI fragments were takenfrom plasmids pBS185 and pBS251, respectively (Example A, herein).

Construction of genes encoding phycocyanin subunit-fusion monomers—X-raycrystallographic data obtained from the highly homologous C-phycocyaninfrom another cyanobacterium Fremyella diplosiphon (42) were used todesign subunit fusions of the Anabaena phycocyanin, see Example A.Straight-line distance between a carbons of the last residue of αsubunit and the first residue of β subunit was measured to be 29.5 Å.The distance between the last residue of β subunit and the first residueof α subunit was 32.6 Å. Although the average sum of three chemicalbonds' length of a peptide unit is 4.3 Å, the actual straight-linedistances between two a carbons were found to range from 3.2 to 3.7 Åamong several peptide units measured in the crystal structure. An11-residue linker was therefore chosen to link the C-terminus of onesubunit to the N-terminus of another. The 11-residue linker issufficiently long to bridge the distance (29.5 to 32.6 Å) between the N-and C-termini of the two subunits, and provides some leeway for bendingshould this be required for higher-order assembly of the resultantfusion phycocyanins.

One obvious difference between phycocyanins of Fremyella diplosiphon andof Anabaena sp. PCC7120 is the lack of the initial Met residue in theAnabaena subunits as a result of posttranslational modification (ExampleA, herein). To reduce possible steric interference in the engineeredAnabaena protein, an Ala was inserted in place of the missing Met at theN-terminus of the subunit following the fusion linker. Since both α andβ subunits have an α-helical C-terminal region made up mostly of smallhydrophobic residues, Ala was chosen as the first residue in the fusionlinker to maintain the local hydrophobic environment. The rest of thelinker peptide (L11) was designed for high structural flexibility, andconsists mostly of Gly residues and two Ser. Incorporation of thesignature site XmnI in the L11 coding sequence not only facilitatescharacterization of PCR products, but also allows easy construction offusions of other proteins to the C-terminus of phycocyanin α or βsubunits (Example C, herein).

Designed oligonucleotide primers incorporating the L11 linker sequencewere used for inverse PCR with template DNA consisting of both pBS185and pBS251 plasmids (Example A, herein), giving plasmids pBS310(NdeI-cpcA-L11-cpcB-EcoRI in pUC19) and pBS307(NdeI-cpcB-L11-cpcA-HindIII). Once sequenced to confirm fidelity, the1047-bp NdeI-EcoRI fragment of pBS310 was cloned into NdeI- andEcoRI-digested pBS150 (Example A, herein), giving plasmid pBS320 whichencodes 6×His-tagged CpcA-L11-CpcB fusion phycocyanin. In like mannerpBS315 was made to encode the 6×His-tagged CpcB-L11-CpcA fusion protein(Table 4). A PCR-generated spontaneous mutant product, in which 6 basesin the signature XmnI site of the L11 linker sequence had been deleted(giving the L9 linker), was also used in this study (pBS319; see Table4).

All other molecular biological manipulations followed standardprocedures (14). To ensure sequence fidelity, all DNA fragments that hadgone through the PCR process (16) were sequenced using the PRISM 373 DNAsequencing system and the dye-terminator cycle-sequencing kit fromApplied Biosystems (Foster City, Calif.). Analyses of DNA and amino-acidsequence data were performed with the programs Editbase (Purdue ResearchFoundation and USDA/ARS) and Lasergene (DNASTAR Inc., Madison, Wis.).

Nomenclature—A recombinant polypeptide encoded by an expression plasmidis referred to by the plasmid number, and the His-tagged proteinfraction isolated by affinity chromatography is designated by theorganism in which the recombinant polypeptide is expressed. For example,a phycocyanin α subunit with both 6×His and Strep2 tags encoded byplasmid pBS327, expressed in E. coli and purified by IMAC is designatedEc327. The purified, characterized protein is then designated asHT-Strep2-α. The same construct expressed in Anabaena sp. PCC7120 isdesignated An327. However, the purified protein is HT-Strep2-α:β,because in Anabaena sp. the HT-Strep2-α forms a stable complex with theholophycocyanin β subunit. Phycobiliproteins expressed in E. coli areapoproteins (lacking attached phycobilins), whereas those expressed inAnabaena sp. are holoproteins, unless otherwise noted.

Design of subunit-fusion phycocyanins—A His-tag at the N-terminus ofeither an α or a β subunit does not interfere with the folding of thesepolypeptides into their native structures (Example A, herein). Twopossible α/β fusions can be generated with an 11-residue (L-11) linker:HTα-L11-β and HTβ-L11-α. In either construct, one subunit, the α or theb, would have a 24-amino acid extension (including the 6×His tag) at theN-terminus and a large fusion, linked through L11, at its C-terminus.Both constructs were made to determine the folding and spectroscopicproperties of such fusions.

Expression of His-tagged phycocyanin β-α fusion protein in E.coli—HTb-L11-α is expressed in E. coli as the apoprotein (lacking thethree phycocyanobilins). At 37° C., in cultures induced with IPTG for 5hrs, the fusion protein (Ec315) represented over 5% of total cellularproteins (>7.5 mg per liter of culture at 10⁹ cells ml⁻¹), and at 30°C., in cultures induced with IPTG for 12 hr, some 20% of total cellularproteins. HTβ-L11-α purified by IMAC could be concentrated up to 0.2 mMprotein (nearly 8 mg m¹⁻¹) in 50 mM Tris-HCl pH 8.0, 50 mM NaCl, 50 mMKCl, 1 mM DTT, and 5% glycerol. The yield and solubility were markedlyhigher than that obtained upon expression of the individual His-taggedphycocyanin subunits, HTα (Ec168) and HTβ (Ec262), in E.coli (Example A,herein). With IPTG induction at 30° C., in the latter cases, yields wereabout 0.1 mg per liter of culture. Upon IPTG induction at 37° C., ˜60%of the individually expressed 6×His-tagged subunits remained in solutionwhile the balance formed inclusion bodies. Thus coexpression of theapo-α and apo-β subunits, inherent in the expression of HTβ-L11-α,evidently promotes folding and concomitant retention of solubility ofthe recombinant apoprotein.

Expression of His-tagged phycocyanin β-α fusion protein in Anabaenasp.—Cultures of Anabaena sp. PCC7120(pBS315) expressing HTβ-L11-αappeared more blue than that of the wild type. Whole-cell absorbancespectra showed a much higher ratio of phycocyanin:chlorophyll a. Despiteappearing very healthy, Anabaena sp. PCC7120(pBS315) cultures grew about30% more slowly than the wild type.

When cell lysate supernatant from Anabaena sp. PCC7120(pBS315) waspassed through a Ni²⁺-NTA affinity column, usually >33% of the totalphycobiliproteins (as estimated from A_(620 nm)) was retained on thecolumn and then eluted with imidazole (resultant protein denoted An315).Such high yields are comparable to that of the strain expressing HTα(>30% of A_(620 nm)), and better than that of the strain expressing HTβ(˜16%) (Example A, herein).

SDS-PAGE analysis showed that the An315 fraction consisted almostentirely of HTβ-L11-α, along with a small amount (<10%) of nativephycocyanin α and β subunits in a ˜1:1 ratio. This finding suggests thatthe fusion construct interacts relatively normally with unmodifiedphycocyanin in the cell. Such interaction to form higher assemblies issupported by analysis of Anabaena sp. PCC7120(pBS315) phycobilisomes,which were indeed found to contain HTβ-L11-α. Much more of the fusionprotein was found in the phycobilisome substructures (rods andhexamers/trimers) than in intact phycobilisomes, suggesting thatphycobilisomes with a higher content of HTβ-L11-α may be less stableduring the preparation procedure. The high amount of HTβ-L11-α relativeto native phycobiliproteins in the hexamer/trimer fraction also suggeststhat the construct is turned over more slowly in the cell. In thiscontext, it is noteworthy that the whole-cell phycobiliproteinfluorescence emission of Anabaena sp. PCC7120(pBS315) was twice as highas that of strains expressing His-tagged α or β subunit, and the growthmedia of this strain often turned blue, the color of phycocyanin.

On SEC-HPLC the An315 preparation fractionated into hexamers and trimersonly, with no monomers observed (Table 5). This result parallels thoseobtained previously with An168 (HTα:β) and An262 (α:HTβ), where the αand β subunits are not covalently linked (Example A, herein). The trimercomponent, (HTβ-11-α)₃, had a λ_(max) at 622 nm at >10⁻⁶ M protein.(HTβ-L11-α)₃ dissociated to monomers at very low protein concentrations,with absorption maxima shifting blue to as low as 615 nm (Table 6), abehavior characteristic of native phycocyanin and of (HTα:β)₃ and(α:HTβ)₃ holoproteins. At high dilution, HTβ-L11-α has anA_(max):A_(360 nm) of 6.2, similar to the values observed for monomericwild-type and His-tagged α:β phycocyanins (Table 6; Example A, herein).The ε for (HTβ-L11-α)₃ at >10⁻⁶ M protein concentration was determinedto be 900,000 M⁻¹ cm⁻¹. The fluorescence emission spectrum for 560 nmexcitation of HTβ-L11-α, with λ at 643 nm and a Φ_(f) of 0.21, werevirtually identical to those of wild-type phycocyanin. The fluorescenceexcitation spectrum for 655 nm emission corresponded well with theabsorbance spectrum (Table 6). The above data show that the HTβ-L11-αfusion phycocyanin has properties virtually identical to those of thewild-type and His-tagged α:β holo-proteins, and indicate that HTβ-L11-αexpressed in Anabaena sp. has a full complement of bilins, i.e., one PCBper α and two per β subunit.

Mass spectral analysis of the SEC-HPLC trimer fraction, (HTβ-L11-β)₃,gave a value of 40,993.2±6.8 which corresponds well to the theoreticalvalue (41,118.8 Dal) of the holoprotein HTβ-L11-α with the initial Metresidue (131.1 Dal) removed. The mass spectral analysis not onlyconfirms the full posttranslational chromophorylation on both the α andβ subunit moieties, but also indicates presence of the posttranslationalmethylation on the γ-amino group of the Asn⁷² residue of the β subunitdomain (51, 52).

Expression of His-tagged phycocyanin α-β fusion protein in Anabaenasp.—Like HTβ-L11-α apoprotein (Ec315), HTα-L11-β (Ec320) was expressedin E. coli in very high yield in soluble form. The phenotype and growthrate of cultures of Anabaena sp. PCC7120(pBS320) expressing HTα-L11-βwere similar to those of strains expressing only His-tagged α or βsubunits. The whole-cell absorbance spectrum of Anabaena sp.PCC7120(pBS320) was similar to that of the wild type but with a slightlyhigher and blue-shifted contribution from phycocyanin, and about 50%higher whole-cell phycobiliprotein fluorescence was observed.

Like HTβ-L11-α (An315), HTα-L11-β (An320) was also expressed at veryhigh yield in Anabaena sp., often accounting for >32% of total cellularphycobiliproteins. SDS-PAGE showed that affinity-purified An320 proteinswere almost entirely HTα-L11-β, with a small amount of copurified nativephycocyanin α and β subunits. The ratio of copurified α to β subunitswas ≧2, suggesting a reduced affinity of HTαX-L11-β for nativephycocyanin β subunits.

On SEC-HPLC, the An320 fraction separates into three components:monomers, trimers, and hexamers. It is noteworthy that nativeC-phycocyanin under the same chromatographic conditions was mainlytrimeric with very little monomer, whereas the HTα-L11-β preparation wasa ˜1:1 mixture of trimers and monomers (Table 5). The theoreticalmolecular weights from these components are 13, 19, and 9% higher,respectively, than the values observed (Table 5). These discrepanciesindicate that the radius of gyration of HTα-L11-β is smaller than thatof the wild-type phycocyanin αβ monomer.

At >10⁻⁶ M protein concentration, the SEC-HPLC trimer and monomerfractions of HTα-L11-β had λ_(max) at 608 and 605 nm, respectively. Thislikely accounts for the blue-shifted phycobiliprotein contribution tothe whole-cell absorbance spectrum of Anabaena sp. PCC7120(pBS320). Likewild-type phycocyanin, the trimer fraction showed a blue shift in itsabsorbance spectrum down to 605 nm with decreasing proteinconcentration, indicative of dissociation of the trimers to monomers. Athigh dilution, HTα-L11-β has an A_(max):A_(360 nm) of 5.2, substantiallylower than the value of 6.4 observed for monomeric wild-type and HTα:βphycocyanins (Table 6; Example A).

The unusual characteristics exhibited by the HTα-L11-β protein purifiedfrom Anabaena sp. were similar to those of apo-HTα:β (Example A,herein). The trimer and monomer SEC fractions of HTα-L11-β weretherefore compared to the HTβ-L11-α holo-protein (both fusion proteinshave identical amino acid composition). When denatured in acid urea, thetrimeric fraction of HTα-L11-β had PCB absorbance about midway betweenthree PCBs per protein (holoprotein) and two PCBs per protein, while themonomeric fraction gave absorbance very close to that of two PCBs perprotein. An interpretation of that result is that the α domain ofHTα-L11-β fusion protein is not fully chromophorylated. Only about 50%of the trimeric HTα-L11-β proteins had PCB in the α domain, while mostmonomeric HTα-L11-β proteins had no PCB addition in the α domain.Because of the nearly equal distribution between trimers and monomers,fewer than 30% of the α subunit domains of affinity-purified HTα-L11-βproteins carry bilin. It is noteworthy that apo-HTα:holo-β andapo-α:holo-HTβ proteins also run mostly as monomers on SEC-HPLC, withapparent molecular weights substantially smaller than theoreticalcalculations (Example A, herein). The fact that most of the HTα-L11-βconstructs have an apo-α domain could also explain why more native αthan native β phycocyanin subunits were copurified with HTα-L11-β, sincethe α-Cys⁸⁴ bilin is involved in the α:β interaction and in stablemonomer packing (55, 62)

Electrospray mass spectral analysis of the SEC-HPLC monomer fraction ofHTα-L11-β gave only one major peak with a value of 40,327.8±5.7,corresponding well with the value of 40,331.7 daltons, obtained bysubtracting from 41,118.8 Dal (molecular weight of holo-HTα-L11-β) thevalues 131.1 (initial Met residue), 57.0 Dal (the C-terminal or thesecond residue from the N-terminus, Gly), 585.0 (one PCB), and 14.0 (onemethyl group). This not only confirms the incomplete bilin addition(likely on the α domain) of HTα-L11-β, but also suggests impairedposttranslational methylation on the γ-amino group of Asn⁷² of the βdomain (51, 52). The latter is unexpected because HTβ (Example A,herein) and HTβ-L11-α (see above) purified from Anabaena sp. are bothfully methylated on β-Asn⁷².

The SEC-HPLC trimer fraction of HTα-L11-β (at >10⁻⁶ M) had a ε of677,000 M⁻¹ cm⁻¹. The fluorescence emission spectrum for 560 nmexcitation, with λ at 643 nm and a Φ_(f) of 0.22, was virtuallyidentical to that of wild-type phycocyanin. The fluorescence excitationspectrum for 655 nm emission corresponded reasonably well with theabsorbance spectrum, with a slight shift in the maximum to 613 nm,likely due to the small amount of native phycocyanin in the preparation(Table 6). The SEC-HPLC monomer fraction of HTα-L11-β had spectroscopicproperties similar to those of apo-HTα:holo-β monomers (Example A).

Effect of shortening the linker—A PCR-generated mutant, HTα-L9-β, withthe linker Ala-(Gly)₄-Ser-(Gly)₃, was isolated in the course ofproducing the HTα-L11-β construct, where L11 isAla-(Gly)₄-Ser-Gly-Ser-(Gly)₃. In other respects the two constructs wereidentical. Like the HTα-L11-β apoprotein (Ec320), the HTα-L9-βapoprotein (Ec319) was expressed in E. coli in high yield. HTα-L9-β(An319) isolated from Anabaena sp. exhibited the same, anomalousspectroscopic properties described above for the HTα-L11-β construct.Analysis of the HTα-L9-β protein by SEC-HPLC showed trimers and monomersin a ratio of 1:4, significantly lower than the ratio of 1:1 observedfor HTα-L11-β. Since the extent of chromophorylation of trimer andmonomer fractions of HTα-L9-β was similar to those of respectivefractions of HTα-L11-β, the reduced trimer formation by HTα-L9-β may bea result of conformation change induced by the shorter linker betweenthe subunits.

Expression of phycocyanin fusion proteins incorporating oligomerizationdomains—The dissociation of phycocyanins to the monomer at low proteinconcentration is highly undesirable when these proteins are used asfluorescent tags, particularly because of the resultant decrease in thenumber of PCB chromophores per tag. The finding that recombinantphycocyanin subunits with a 24-residue extension at the N-terminus foldand assemble in the same manner as native phycocyanin subunits (ExampleA, herein) suggested the introduction of the GCN4 oligomerizationdomains (60) at the subunit N-termini to produce stable phycocyaninoligomers. Expression of phycocyanin α subunit fused at the N-terminusto the trimerization domain GCN4-pII—The 33-residue peptide GCN4-pIIforms homotrimeric parallel coiled coils with K_(D)<10⁻⁹ M (60). PlasmidpBS314 (encoding HT-pII-α) was constructed to express a GCN4-pII-CpcAfusion (Table 4).

When HT-pII-α was expressed in E.coli, the recombinant protein was foundalmost entirely in inclusion bodies. Similar results were observedwhether the induction was at 30 or 37° C. This behavior is in sharpcontrast to that seen with HTα [Ec168; (Example A, herein)], where mostof the recombinant protein remains soluble. A likely explanation is thattrimerization of the GCN4-pII in the fusion protein is faster than thefolding of the phycocyanin subunit domain and interferes with folding ofthe latter by promoting random aggregation of the partially foldedsubunit domains.

Cultures of Anabaena sp. PCC7120(pBS314) expressing HT-pII-α showed nonegative phenotype, but had a bluer appearance than the wild type.Whole-cell absorbance spectra of Anabaena sp. PCC7120(pBS314) showed ahigher ratio of phycocyanin:chlorophyll a. Whole-cell fluorescence was˜70% higher than that of the wild type or cells expressing HTα. Sucrosedensity gradient fractionation of Anabaena sp. PCC7120(pBS314)phycobilisome preparations showed that HT-pII-α represented a muchhigher proportion of the total phycocyanin in the trimer/hexamerfraction than in the intact phycobilisome fraction.

Over 40% of the phycobiliprotein in Anabaena sp. PCC7120(pBS314) celllysate supernatant bound to the Ni²⁺-NTA column and eluted with 200 mMimidazole. This was the highest relative level of recombinantphycobiliprotein of any of the His-tagged constructs we have expressedin the course of these studies. An314 bound much more tightly to theNi²⁺-NTA column than His-tagged phycocyanin constructs not containingoligomerization domains. The tightness of binding was evidenced by theslow desorption of the recombinant protein from the Ni²⁺-NTA resin andthe larger volume of imidazole buffer required for elution. Suchbehavior is to be anticipated from a molecule in which threepolypeptides, each with a 6×His tag, are trimerized through the GCN4-pIIcoiled coil. SDS-PAGE analysis showed that the An314 phycobiliproteinfraction was a stoichiometric HT-pII-α:β holoprotein complex.Chromatography on SEC-HPLC showed that the HT-pII-α:β preparationwas >75% trimer with some higher molecular weight component(s),presumably hexamer. Monomers were not detected.

The spectroscopic properties of (HT-pII-α:β)₃ are compared with those ofthe native C-phycocyanin trimer, (α:β)₃ in Table 6. The trimerizedconstruct has significantly higher A_(max):A_(360 nm) ratio than thenative (α:β)₃ and a higher Φ_(F). Moreover, the absorbance spectrum of(HT-pII-α:β)₃ was unchanged at low protein concentrations where nativephycocyanin, HTα:β, and α:HTβ were monomeric with significantlyblue-shifted absorbance maxima and decreased A_(max):A_(360 nm) ratios(Table 6; Example A, herein). Fluorescence polarization measurementsprovided an independent assessment of the stability of (HT-pII-α:β)₃ atlow protein concentrations. In trimeric phycocyanin, the fluorescencepolarization is very low because of rapid energy transfer among the ninephycocyanobilin chromophores. In contrast, in the phycocyanin monomer,the three PCBs are well separated and depolarization through energytransfer is minimized. Our data show that at high proteinconcentrations, where various phycocyanin preparations are trimeric,their fluorescence polarization is similar and low, with values of 0.035to 0.070. The fluorescence polarization of HTα:β and α:HTβ, like that ofnative phycocyanin, rose sharply at very low protein concentrations,indicative of trimer dissociation, whereas that of (HT-pII-α:β)₃ wasindependent of protein concentration.

Expression of phycocyanin α subunit fused at the N-terminus to thetetramerization domain GCN4-pLI—The 33-residue peptide GCN4-pLI formsvery stable homotetrameric parallel coiled coils (60). Several plasmidswere constructed to express GCN4-pLI-α fusion proteins (Table 4),including plasmids pBS321 (encoding HT-pLI-α) and pBS323 (encodingHT-Strep2-pLI-α). The 10-residue Strep2 tag was incorporated into theHT-Strep2-pLI-α to test whether it would confer streptavidin-bindingspecificity on the construct.

When expressed in E. coli, GCN4-pLI-α fusion proteins, Ec321 (HT-pLI-α)and Ec323 (HT-Strep2-pLI-α), were mostly found in inclusion bodies. Incontrast, when expressed in Anabaena sp. the fusion proteins An321 andAn323 remained soluble. The yields of these His-tagged phycobiliproteinsfrom this strain were high, reaching >20% of total phycobiliprotein. Asdescribed above for the GCN4-pII-α fusion proteins, the GCN4-pLI-αfusion protein (An321) bound very tightly to the Ni²⁺-NTA resin.SDS-PAGE analysis showed that An321 protein fraction purified by IMAChad the composition of HT-pLI-α:β. Analysis by SEC-HPLC showed twocomponents, a small amount of tetramer, (HT-pLI-α:β)₄, and the balanceas a much larger component. The elution position of the latter layoutside the range of calibration of the size standards for the column,but extrapolation of the, calibration curve allowed an estimate of thesize of the larger component. The calculated molecular weight wasconsistent with that of a trimer of tetramers (Table 5).

Presumably, such a trimer would be formed by the interaction of three(HT-pLI-α:β)₄ assemblies through one of their phycocyanin α:β domains(while the other three α:β domains forming a trimer) in a manneranalogous to that leading to the formation of (HT-pII-α:β)₃. In such a“trimer of tetramers”, all α:β monomers are within phycocyanin trimers.Tetramers, each held together by a GCN4-pLI domain, would be theexpected products of dissociation of such a “trimer of tetramers” atvery low protein concentration. In accord with this expectation, theabsorbance spectrum of HT-pLI-α:β at low protein concentration showed aλ_(max) of 621 nm and an A_(max):A_(360 nm) ratio of 7.5 (Table 6),values very similar to those obtained for (HT-pII-(α:β)₃ (see above).

HT-Strep2-pLI-α:β (An323) exhibited physical and spectroscopicproperties identical to those of HT-pLI-α:β (An321) (Table 6), showingthat the presence of the Strep2 sequence does not interfere withtetramerization by the GCN4-pLI domain.

Expression of subunit-fusion phycocyanins fused at the N-terminus to thetrimerization domain GCN4-pII—As described above, His-tagged β-αsubunit-fusion phycocyanin (An315) has spectroscopic propertiesidentical to those of HTα:β and α:HTβ. Like those proteins, however,HT-β-L11-α also dissociates to monomers at high dilution. To increaseutility of the HT-β-L11-α fusion phycocyanin as a fluorescent label, wefused the GCN4-pII domain to this construct to enhance trimer stability.

Plasmid pBS358 encodes HT-pII-β-L11-α (Table 4). When expressed in E.coli, HT-pII-β-L11-α apoprotein was found mostly in inclusion bodies,but when expressed in Anabaena sp. the HT-pII-β-L11-α protein remainedsoluble. Cultures of Anabaena sp. PCC7120(pBS358) had phenotypes verysimilar to those of the Anabaena sp. PCC7120(pBS315) (see above),including the slower growth, “bluer” color, and increased whole-cellfluorescence. Analysis of phycobilisome preparations from this strainshowed that HT-pII-β-L11-α was incorporated into the phycobilisomes. The6×His-tagged phycobiliprotein fraction (An358) purified by IMACrepresented >22% of total cellular phycobiliproteins. As noted above forAn314 (HT-pII-α:β), the An358 fraction also bound very tightly to theNi2+-NTA resin.

SDS-PAGE analysis of the An358 fraction showed that it consisted >90% ofHT-pII-β-L11-α along with a small amount of copurified wild-typephycocyanin α and β subunits (in a ratio of 1:1). Analysis by SEC-HPLCat protein concentrations <500 μg/ml, showed that HT-pII-β-L11-αpreparation was largely trimeric with a small amount of a largercomponent (Table 5). Comparison of the spectroscopic properties of(HT-pII-β-L11-)3, purified by SEC-HPLC, with those of nativeC-phycocyanin trimer (αβ)3, showed no significant differences (Table 6).The absorbance spectrum of (HT-pII-b-L11-a)3 did not change withdilution to very low protein concentration (Table 6), indicating thatthe GCN4-pII domain prevented dissociation of the trimer.

The α-β fusion constructs purified from Anabaena sp., HTα-L9-β andHTα-L11-β, preferentially form monomers even at micromolar proteinconcentrations (see above). The similar construct incorporating theGCN4-pII domain, HT-pII-α-L11-β, is encoded by plasmid pBS362 (Table 4).The HT-pII-α-L11-β protein purified from Anabaena sp., An362, was foundto be largely trimeric (84%) with the remainder hexameric (Table 5). Nomonomer was observed in the SEC-HPLC analysis. It is evident that thetrimeric state of HT-pII-α-L11-β depends on trimerization of theGCN4-pII domain. Although remaining trimeric at low proteinconcentrations, the HT-pII-α-L11-β protein had spectroscopic propertiessimilar to those of HTα-L11-β protein (Table 6), including theincomplete chromophorylation of the a domain.

Expression of recombinant phycocyanins with a tag conferringbiospecificity—The constructs described above successfully address therequirement that the phycocyanin constructs maintain their oligomericstate at very low proteins concentrations. Next, we examine theintroduction of an additional domain into such constructs, one whichconfers biospecific recognition. As a test case, we introduced a tagwhich binds specifically to streptavidin. Recombinant phycocyaninscontaining such a tag are immediately useful as reagents in the manyapplications of the widely used streptavidin-biotin methodologies (63).

Expression of Strep2-tagged phycocyanin constructs—The 10-residue Strep2peptide forms complex with streptavidin with a K_(D) of 7.2×10⁻⁵ M (57).It is believed that the Step2 tag retains its affinity for streptavidinwhen fused either to an N- or C-terminus of a protein (57). PlasmidspBS327 and pBS323 were constructed to encode HT-Strep2-α andHT-Strep2-pLI-α, respectively (Table 4). E. coli and Anabaena strainsexpressing either fusion protein were phenotypically identical to thoseexpressing the corresponding non-Strep2-tagged proteins.Affinity-purified HT-Strep2-α:β (An327) and HT-Strep2-pLI-α:β (An323)proteins also had spectroscopic and aggregation properties very similarto corresponding non-Strep2-tagged proteins HTα:β (An168) and HT-pLI-α:β(An321) (Tables 5 and 6).

When blotted on a membrane, HT-Strep2-α (Ec327 and An327 andHT-Strep2-pLI-α (Ec323 and An323) were readily detected by probing withstreptavidin-alkaline phosphatase conjugate, indicating that the Strep2peptide sandwiched between the 6×His tag and the GCN4-pLI domain stillretains its affinity for streptavidin. When streptavidin-coated agarosebeads were used as target, HT-Strep2-α:β holoprotein was largely removedfrom the beads on washing, a result anticipated from the low affinity ofa single Strep2 tag for streptavidin [K_(D)=7.2×10⁻⁵ M; (57)]. TheHT-Strep2-pLI-α:β holoprotein, with its multiple Strep2 tags, bound muchmore strongly to the streptavidin-coated beads. This experimentdemonstrates that oligomeric phycobiliprotein fusions with multipleStrep2 tags show the appropriate target specificity instreptavidin-based fluorescence assays.

Example C. Recombinant Phycobiliprotein Fusion Proteins WithCarboxyl-terminal Tagging

We present here the in vivo production of biotinylated phycocyaninconstructs that are readily usable in the many well-developedbiotin/avidin applications (63). Materials and methods essentially asdescribed in Example A or B are not restated.

Construction of expression plasmids—Plasmids coding for relevant proteinfusion constructs are listed in Table 7. The expression vector pBS150v[(Example A); GenBank Accession number AF177932] was used to makeplasmid pBS339v (4,677 bp) for expression of His-tagged proteins capableof being biotinylated in E. coli. The 7 codons between the 6×His tag andthe TEV site in pBS150v were replaced with the 21 codons specifying theBTN tag. The 13-residue sequence of the BTN tag was derived from theconsensus sequence that can be biotinylated in E. coli (64). An AsuIIrestriction site was designed into the BTN sequence as a signature siteto facilitate PCR product characterization and subsequent recognition ofthe BTN tag sequence. The NdeI-cpcA-HindIII fragment from pBS185(Example A) was inserted between NdeI and HindIII sites of pBS339v,giving plasmid pBS329v (5,138 bp) encoding 6×His- and BTN-taggedphycocyanin α subunit (Table 7). The BTN tag is positioned on thecarboxyl-terminal side of the 6×His tag, so that any recombinantmolecules that have lost the biospecificity tag through proteolysis arenot retained on the IMAC column. The NdeI-glbN-HindIII fragment fromplasmid pGlbN (66) was similarly cloned to give plasmid pBS355, encoding6×His- and BTN-tagged cyanoglobin (Table 7). The same NdeI-HindIIfragment cloned into pBS150v gave plasmid pBS121v (5,005 bp) encoding6×His-tagged cyanoglobin (Table 7).

The cloning vector pBS370v (4,589 bp) for expression of C-terminal Streptagged proteins (Table 7) was derived from the cloning vector pBS152v[(Example A); GenBank accession No. AF177933] using inverse PCR. The45-bp HindIII-BglI fragment at the end of the multiple cloning sites wasreplaced with the 68-bp HindIII-Strep-BglI sequence. An EheI site wasengineered into the Strep tag coding sequence (56) as a signature site.Phycocyanin genes, in the form of NdeI-XmnI fragments from cognateplasmids (Example B) were inserted between NdeI and XmnI sites ofpBS370v to generate phycocyanin-Strep tag fusions. The expressioncloning vector pBS350v, an enhanced version of pBS152v, was created byreplacing bp 3,801 to 4,070 of pBS152v with the 333-bp sequencedescribed above.

DNA fragment encoding the 114-AA C-terminal portion of the Anabaena sp.PCC7120 BCCP protein (BCCP114) was amplified from Anabaena genomic DNA.The 0.36-kb PCR fragment was digested with EcoT22I and cloned into theEcoT22I site of pBS350v, giving plasmid pBS344v (4,977 bp). Again,phycocyanin genes, in the form of NdeI-XmnI fragments from cognateplasmids (Example B), were inserted between NdeI and XmnI sites ofpBS344v to generate phycocyanin-BCCP114 fusion constructs with the BTNflexible linker. The 20-residue flexible linker between the two moietieswas designed to (a) bear a thrombin recognition and cleavage site toallow separation of the fusion partners if so desired, and (b) besufficiently long to allow packing of BCCP114 on the outside of the rodsubstructures after the fusion protein is assembled in phycobilisomes(Example A, Example B). The GCN4-pII trimerization domain was introducedvia modular cloning (Example B) between the 6×His tag and the TEVprotease site N′ of the phycocyanin genes.

Expression of BTN-tagged fusion proteins—The 13-residue BTN tag has theconsensus sequence of peptides found to be biotinylated in E. coli (64).Covalent attachment of a biotin to the Lys residue is apparentlycatalyzed by the biotin ligase encoded by the birA gene (65, 67). TheBTN tag was fused to the N-terminus of the phycocyanin α subunit in anattempt to produce in vivo biotinylated phycocyanin. When the HT-BTN-αphycocyanin subunit, encoded by plasmid pBS329, was expressed in E.coli, the purified Ec329 protein was shown biotinylated in Westernanalysis. The same protein produced in Anabaena sp. PCC7120 (An329),however, was very poorly biotinylated, if at all, even when the culturemedium had been supplemented with as much as 500 μM biotin. Two otherassays for the presence of biotin on An329, competition with HABA{2-[(4′-hydroxyphenyl)-azo]benzoic acid} for streptavidin in solution(68) and mobility shift in SEC-HPLC, indicated lack of biotinylation.Parallel results were obtained in a comparison of Ec317 (HT-BTN-pII-α)and An317 (HT-BTN-pII-α:β) proteins.

Sucrose density gradient experiments showed that both HT-BTN-α (An329)and HT-BTN-pII-α (An317) proteins were assembled into thephycobilisomes. To test whether the lack of An317 and An329biotinylation in vivo was due to the fact that the recombinantphycocyanins were quickly assembled into the phycobilisome uponbiosynthesis and therefore not available to the biotin ligase, thecyanoglobin (GlbN) protein from cyanobacterium Nostoc commune (66) wasused. This myoglobin-like monomeric hemoprotein has a specificsubcellular location around the periphery of the cytosolic face of thecell membrane (69), but is not membrane-bound. His-tagged cyanoglobinsHT-GlbN and HT-BTN-GlbN (encoded by plasmids pBS121 and pBS355,respectively; Table 7) expressed in both E. coli and Anabaena sp. werefound to have absorbance spectra nearly identical to that described forthe native GlbN (70). However, while the Ec355 protein was found to bebiotinylated, the An355 protein was not. The negative results forbiotinylation in Anabaena sp. of all three different proteins tested,An317, An329, and An355, suggest that the BTN tag is not recognized bythe Anabaena biotin ligase.

Analysis of the IMAC-purified An329 proteins by SDS-PAGE incated thecomposition HT-BTN-α:β. The An329 SEC-HPLC elution profile andspectroscopic characteristics were virtually identical to thenon-BTN-tagged counterpart An168 (HTα:β; Table 8). The yield, however,was surprisingly low: An329 recovered by IMAC was <3% of total cellularphycobiliprotein, over 10-fold less than obtained for An168. This wasalso the case for An317 compared to its non-BTN-tagged counterpart An314[HT-pII-α:β; (Example B)]. The yield of An355 was also dramaticallylower than that of An121. These results suggest that the BTN tagdestabilizes the tag-bearing proteins in Anabaena cells. Since An317 andAn329 are assembled into the phycobilisomes, the latter may also bedestabilized. Higher turnover of phycobilisomes and of An329 proteins inthe cell may explain the observed phenotype of lower cellularphycobiliproteins in the strains Anabaena sp. PCC7120(pBS317) andAnabaena sp. PCC7120(pBS329).

Expression of truncated Anabaena BCCP114 protein—Since the BTN tag wasnot biotinylated in Anabaena sp., naturally occurring biotinylatedproteins were investigated. A truncated gene encoding the C-terminal 114residues of the Anabaena BCCP protein, covering the biotinylation domain(corresponding to the E. coli BCCP84 that is sufficient forenzyme-catalyzed biotinylation) and a large portion of the flexiblelinker, was amplified from the genome and cloned into pBS350, givingplasmid pBS344 (Table 7). Plasmid pBS344 encodes the 6×His-taggedAnabaena BCCP114 with a long linker bearing the TEV and thrombinprotease sites.

E. coli cultures expressing the 6×His-tagged Anabaena BCCP114 protein(Ec344) grew normally, and produced very soluble Ec344 protein at >10 mgper liter of culture grown and induced at 37° C. The protein was shownbiotinylated by Western analysis. Electrospray mass spectroscopyanalysis suggested that about 20% of the Ec344 protein produced underthese particular conditions was biotinylated. Growth and induction at30° C. for longer period of time was attempted to increase thepercentage of biotinylated holoprotein fraction of purified Ec344.However under these conditions, the yield of affinity purified proteinwas greatly decreased, possibly as a result of lowered production andincreased degradation of the Ec344 protein.

Cultures of Anabaena sp. PCC7120(pBS344) expressing the An344 proteinwere fairly healthy, albeit having a slightly reduced level of totalcellular phycobiliproteins. The yield of An344 protein, however, wasquite low (usually <1 mg per liter of dense culture), possibly as aresult of proteolytic clipping of the relatively long linker between the6×His tag and the BCCP114 domain. The An344 protein was shown to bebiotinylated in Western analysis. Electrospray mass spectral analysisindicated that about 40% of the molecules were holo- (i.e.,biotinylated) proteins, substantially higher than the fraction of thoseproduced in E. coli. These results encouraged the study of C-terminalphycocyanin α and β fusions with BCCP114.

Expression of phycocyanin β subunit-BCCP114 fusion proteins—PlasmidpBS353 encodes the fusion protein HTβ-BCCP114 (Table 7). Overexpressionof Ec353 in E. coli gave high yield of the protein in soluble form.Since expression of Ec262 (HTβ) under similar conditions leads tosubstantial formation of inclusion bodies (Example A), the BCCP114domain appears to enhance the solubility and folding of the fusionprotein. The HTβ-BCCP114 preparation from E. coli was found biotinylatedin Western analysis.

Cultures of Anabaena sp. PCC7120(pBS353) generally had an unhealthy,yellowish green appearance, and the growth rate was at least 20% slowerthan that of the wild type even under high-light conditions. Theyellowish green color of the cultures results from ˜50% reduction ofcellular phycocyanin as indicated by whole-cell absorption spectra. UponIPTG induction of overproduction of the HTβ-BCCP114 protein, cultures inlate-log growth frequently stopped growing, and further incubation ofthe culture often led to general cell lysis. This unexpected result,which was also observed with the HTα-BCCP114 construct (An351; seebelow), led to the abandonment of IPTG induction of Anabaena sp.PCC7120(pBS353) cultures. Expression of the HTβ-BCCP114 protein,therefore, was left to the leaky trc promoter (Example A), and generallyresulted in a fairly low yield of An353 (<5% of total cellularphycobiliproteins). Nonetheless, very high cell densities could beobtained with these uninduced cultures.

The HTβ-BCCP114 protein was incorporated into phycobilisomes, with noobvious destabilizing effects. On SDS-PAGE analysis, however, theaffinity-purified An353 protein had a substantially larger amount of theHTβ-BCCP114 subunit than the partner phycocyanin holo-α subunit,indicating a lowered stability of α:HTβ-BCCP114 as compared with that ofα:HTβ. Also, a small fraction of the HTβ-BCCP114 preparation had lostthe BCCP114 moiety by proteolysis.

On SEC-HPLC the An353 preparation separated into two major components.The larger component, with a retention time of about 12.4 min, had anapparent mass of 183.7 kDal, somewhat larger than 162.2 kDal calculatedfor the trimer, (α:HTβ-BCCP114)₃. On SDS-PAGE this larger fraction hadonly phycocyanin α and HTβ-BCCP114 subunits, in nearly 1:1 ratio. Uponexposure to Zn²⁺, fluorescence from the HTβ-BCCP114 band under UVillumination was about twice as bright as that from the phycocyaninholo-α subunit, suggesting full bilin content (two PCBs per β) of therecombinant phycocyanin β subunit. The smaller component, eluted at˜14.4 min, contained mostly HTβ-BCCP114, with an apparent molecularweight corresponding to that of the homodimer, (HTβ-BCCP114)₂. Whilethis result is in accord with the observation that phycocyanin HTβsubunits can form stable homodimers (Example A), it also suggests thatthe HTβ-BCCP114 subunits have a lowered affinity for the phycocyanin αsubunit, and may lose some of the α subunits during affinitypurification.

Polypeptides in the HTβ-BCCP114 preparation lacking the BCCP114 portionrepresent a higher proportion of the fraction containing (HTβ-BCCP114)₂than of the (α:HTβ-BCCP114)₃ fraction. Such proteolytic removal of theBCCP114 domain of Ec353 was not observed in E. coli. The degradationobserved in the preparations from Anabaena cells may therefore be theresult of the much longer time the fusion protein An353 remains insideAnabaena cells, and may also reflect higher activity of Anabaena sp.proteases towards the long, thrombin site-bearing linker.

Spectroscopic properties of the trimeric fraction, (α:HTβ-BCCP114)₃,were similar to those of (α:HTβ)₃ (Table 8). The slightly blue-shiftedλmax of (α:HTβ-BCCP114)₃ and the lower A_(max):A_(360 nm) ratio mayreflect incomplete bilin addition to the β subunit, with a consequentperturbed conformation and lowered trimer stability in a portion of thepreparation.

Western analysis showed that HTβ-BCCP114 produced in Anabaena sp. wasbiotinylated. Binding experiments with monomeric avidin-coated beadswere performed to further assess the utility of An353 proteins aslabels. When avidin molecules were in excess, ˜30% of the An353preparation could be immobilized on the beads and then specificallyeluted, indicating a level of HTβ-BCCP114 biotinylation in Anabaena sp.of about 30%. When an excess of An353 was used, saturation by therecombinant phycocyanin of nearly all of the biotin-binding sites on theavidin-coated beads was achieved. With appropriate excitation, thestained beads emitted brilliant red fluorescence.

Expression of HTβ-BCCP114 fusion protein bearing the GCN4-pIItrimerization domain—Only about a half of the HTβ-BCCP114 expressed inAnabaena retained the phycocyanin α subunit during purification. We hadnoted in earlier studies (Example B) that phycocyanin fusion proteinswith GCN4-pII trimerization domains remained trimeric even at very lowprotein concentrations and contained both subunits in stoichiometricamounts. This is most likely attributable to the greatly increased localconcentration of subunits.

HT-pII-β-BCCP114, encoded by plasmid pBS359 (Table 7), was expressed athigh level in E. coli. About 25% of Ec359 could be isolated in solubleform, much more than seen with pII-phycocyanin subunit fusion constructs(Example B), again showing the increase in solubility attributable tothe presence of the BCCP114 domain (see above).

Cultures of Anabaena sp. PCC7120(pBS359) were yellowish, similar tothose of Anabaena sp. PCC7120(pBS353), with an even slower growth rate.However, such cultures grew to very high cell densities after IPTGinduction.

The HT-pII-β-BCCP114 protein was assembled into phycobilisomes with noobvious destabilizing effect. In contrast to An353, affinity-purifiedAn359 protein preparation had the HT-pII-β-BCCP114 subunit and thephycocyanin holo-α subunit in a 1:1 ratio. The yield of An359 wassurprisingly low, generally <2% of total cellular phycobiliproteins.Almost no HT-pII-β lacking the BCCP114 moiety was observed, suggestingthat in the stable trimers, (α:HT-pII-β-BCCP114)₃, the long linkerbetween β and BCCP114 is shielded from proteases.

On SEC-HPLC, the An359 protein preparation migrates as a component withan apparent mass of 475.2 kDal, significantly higher than the 350.3 kDalcalculated for a hexamer, [(α:HT-pII-β-BCCP114)₃]₂. A phycocyaninhexamer is normally formed by face-to-face stacking of two trimers (42).A model of the (α:HT-pII-β-BCCP114)₃ trimer reveals that a hexamer couldform by two trimers stacking on the BCCP114 face, with the BCCP114domains on the outside of the trimer rings. Such a hexamer would have alarger radius of gyration and exhibit a higher apparent mass on SEC. Itshould be noted that at very low protein concentration the hexamer isexpected to dissociate into trimers. Spectroscopic properties of(α:HTβ-BCCP114)₃ (Table 8), were very similar to those ofGCN4-pII-bearing constructs such as (HT-pII-α:β)₃.

Western analysis showed that HT-pII-β-BCCP114 obtained from Anabaena sp.was biotinylated. The utility of this construct as a fluorescent labelwas explored in binding experiments with avidin-coated beads. Withavidin in excess, about 75% of (α:HT-pII-β-BCCP114)₃ was immobilized onthe beads and then specifically eluted off. The extent of biotinylationof the BCCP114 domain in HT-pII-β-BCCP114 proteins is presumably ˜30%,similar to that seen in HTβ-BCCP114 (An353). The higher bindingpercentage is anticipated, since only one of the three BCCP114 domainsneeds to be biotinylated for the entire trimer to bind to the beads.When an excess amount of (α:HT-pII-β-BCCP114)₃ was used, the amount ofα:HT-pII-β-BCCP114 monomer equivalents immobilized was nearly 2.5-foldthat of monomeric avidins. Under phycocyanin excitation,(α:HT-pII-β-BCCP114)₃-stained avidin- and streptavidin-coated beads werehighly fluorescent.

Expression of phycocyanin-BCCP114 fusion protein with covalently bridgedα and β subunits—Constructs with covalently bridged α and β subunitsprovide a way of ensuring 1:1 α:β stoichiometry, as demonstrated earlierwith the HTα-L11-β construct (Example B). HTα-L11-β-BCCP114 encoded byplasmid pBS361 (Table 7) was expressed well in E. coli and acceptably inAnabaena sp.

HTα-L11-β-BCCP114 protein was assembled into the phycobilisome. As withHTα-L11-β (Example B), such phycobilisomes were less stable. Theaffinity-purified An361 fraction represented ˜10% of total cellularphycobiliprotein and consisted >90% of HTα-L11-β-BCCP114. A small amountof native phycocyanin α and β subunits copurified withHTα-L11-β-BCCP114, indicating that the α and β subunits in the fusionconstruct retained their ability to interact with native subunits. Asmall fraction of the HTα-L11-β-BCCP114 proteins had lost the BCCP114portion by proteolysis.

SEC-HPLC fractionation showed that the An361 preparation consisted oftrimers and monomers in an ˜1:1 ratio. Spectroscopic properties of thetrimers, (HTα-L11-β-BCCP114)₃, were found very similar to those of(HTα-L11-β)₃ (Table 8), indicating that carboxyl terminal attachment toBCCP114 did not perturb the phycocyanin subunit domains. In a relatedconstruct, involving a carboxyl terminal fusion of the α subunit,(HTα-L11-β)₃, bilin addition to the α subunit domain was shown to bevery incomplete (Example B). Since (HTα-L11-β)₃ and (HTα-L11-β-BCCP114)₃have virtually identical spectroscopic properties (Table 8), bilinaddition to the α subunit domain of (HTα-L11-β-BCCP114)₃ is presumablyalso incomplete.

The HTα-L11-β-BCCP114 protein preparation from Anabaena sp. was shown tobe biotinylated in Western analysis. In binding experiments withstreptavidin- and avidin-coated beads, the An361 preparation gaveresults similar to those obtained with HTβ-BCCP114 (see above),indicating ˜30% biotinylation of the BCCP domain of HTα-L11-β-BCCP114.

Expression of phycocyanin-BCCP114 fusion protein with covalently bridgedα and β subunits and the GCN4-pII trimerization domain—Plasmid pBS365was constructed to encode the HT-pII-α-L11-β-BCCP114 (Table 7). UnlikeEc362 (HT-pII-α-L11-β) which is found almost entirely in inclusionbodies (Example B), Ec365 expressed in E. coli remained soluble, againindicating the solubility-promoting effect of the BCCP114 domain.

HT-pII-α-L11-β-BCCP114, expressed in Anabaena sp. PCC7120(pBS365), wasfound to be assembled into the phycobilisome. The yield ofaffinity-purified An365 was >5% of total cellular phycobiliprotein. TheAn365 preparation consisted of >90% HT-pII-α-L11-β-BCCP114 with a smallamount of copurifying native phycocyanin α and β subunits. Virtually noloss of the BCCP114 moiety was observed.

On SEC-HPLC, the An365 preparation was found to consist mostly ofhexamers, with a small fraction of trimers. (HT-pII-α-L11-β-BCCP114)₃had spectroscopic properties similar to those of (HT-pII-α-L11-β)₃(Table 8), again consistent with incomplete bilin addition to thephycocyanin α domain (Example B).

The An365 from Anabaena sp. was shown to be biotinylated in Westernanalysis and gave results in binding experiments with monomericavidin-coated beads quantitatively similar to those obtained with An359,(α:HT-pII-β-BCCP114)₃. Streptavidin-coated beads stained with excess ofAn365 appeared blue and emitted brilliant red fluorescence underappropriate excitation.

Expression of phycocyanin α subunit-BCCP114 fusion proteins—Recombinantphycocyanin α subunits, with the amino-terminus fused to polypeptides ofvarying length, display unmodified bilin content and spectroscopicproperties (Example A, Example B). As noted above, fusions at thecarboxyl terminus of the α subunit interfered with bilin addition. Toexamine whether this behavior is specific to particular C-terminalfusions or is general, four phycocyanin α-BCCP114 fusion constructsanalogous to the β-BCCP fusions described above were prepared (Table 7).

Expression of the four constructs, Ec351, Ec357, Ec360, and Ec364, in E.coli gave results very similar to those observed with the fourcorresponding phycocyanin β-BCCP114 constructs (see above). Expressionof the four α-BCCP114 constructs in Anabaena sp., however, gave verydifferent results.

An351 (HTα-BCCP114) represented <0.5% of total cellularphycobiliprotein. SDS-PAGE showed that the HTα-BCCP114 polypeptide had avery low bilin content, indicating that the BCCP114 extension on the αsubunit greatly interferes with bilin addition. The low yield is likelyattributable to the ease of proteolysis of the apo-HTα-BCCP114polypeptide. The An351 preparation contained little phycocyanin βsubunit, suggesting reduced affinity to the apo-HTα-BCCP114. Theproteolytic cleavage of the long peptide linker between α and BCCP114was also high in the apo-HTα-BCCP114 polypeptide. An351 preparationsoften contained a substantial amount of degradation product ofHTα-BCCP114 in which the BCCP114 moiety had been clipped off. Theextremely low yield of An351 also suggests that the protein is rapidlyturned over in the cell.

Results from expression of An357 (Table 7) were nearly identical tothose from An351, except that the native phycocyanin β subunit wascopurified with the (mostly apo-) HT-pII-α-BCCP114 subunit in nearly 1:1ratio. Results from expression of An360 (HTβ-L11-α-BCCP114) and An364(HT-pII-β-L11-α-BCCP114) were very similar. The yield was ˜5% of totalcellular phycobiliprotein, substantially higher than that obtained fromAn351 and An357. A large percentage of the purified fusion protein,however, had lost the BCCP114 moiety. These results showed thatinterference with bilin addition to the phycocyanin α-subunit domain wasa feature common to all of these different BCCP114 fusion constructs.

Expression of phycocyanin α subunit-Strep tag fusion proteins—To addressthe question of the possible dependence of bilin addition to thephycocyanin α subunit on the size of the carboxyl-terminal fusionpartner, such fusions were prepared with the 10-residue Strep tag (Table7).

The yield of the His-tagged protein preparation, An386, from Anabaenasp. PCC7120(pBS386) expressing HTα-Strep was <1% of total cellularphycobiliprotein (in great contrast to >30% usually obtained from cellsexpressing HTα). On SEC-HPLC the An386 preparation runs almost entirelyas monomer, with a very small amount of trimers. The monomer peakcontained HTα-Strep and native phycocyanin β in nearly 1:1 ratio. Whilethe β subunit was the normal holoprotein, the HTα-Strep subunits werealmost entirely apo-subunits. The apo-HTα-Strep appeared to have loweredaffinity for the phycocyanin β subunit, as indicated by the observationthat fractions collected from the trailing part of the monomer peak onSEC-HPLC were enriched in the apo-HTα-Strep subunit relative to theholo-β subunit. The spectroscopic properties of the HTα-Strep:β monomerwere very similar to those of the apo-HTα:holo-β monomer (Table 8;Example A), including the characteristic broad peak in the fluorescenceexcitation spectra. Thus, even a short extension on the carboxylterminus of phycocyanin α subunit affects the posttranslational bilinaddition.

Expression of phycocyanin fusion protein with covalently bridged α and βsubunits and the Strep tag—In a final attempt to address the problem ofincomplete bilin addition in carboxyl terminal fusions of thephycocyanin α-subunit, we examined behavior of HTβ-L11-α-Strep fusions(encoded by plasmid pBS394; Table 7). The yield of affinity-purifiedAn394 was ˜10% of total cellular phycobiliprotein. Unlike the An315preparation (from cells expressing HTβ-L11-α) that consists mostly oftrimers, the An394 proteins is mostly monomeric, with small amounts oftrimeric and hexameric components. Proteins in the higher assemblystates had a higher bilin content, with those in the hexamer fractionapproaching three PCBs per HTβ-L11-α-Strep molecule, i.e., holoproteins.Proteins in the monomer fraction on average contained two PCBs perHTβ-L11-α-Strep molecule, most likely lacking the α subunit bilin. Intotal, <10% of the polypeptides in the An394 preparation carried bilinon the α subunit domain, a small but significant improvement over theHTα-Strep:β construct. Most An394 preparation could be immobilized onstreptavidin-coated beads, showing that the Strep tag in theHTβ-L11-α-Strep molecules has retained its affinity for streptavidin andis available for binding.

TABLE 1 Properties of Anabaena sp. PCC712O and derivative strains and ofexpression vectors carried by these strains relevant to the productionof His-tagged C-phycocyanin α and β subunits Strain genomic Polypeptideconstruct Strain and vector^(a) characteristics encoded on vectorPCC7120(pBS168) Wild-type HTα PCC7120(pBS262) Wild-type HTβ B646(pBS168)cpcBAC^(b) has a transposon HTα inserted between the promoter and thecpcB open reading frame B646(pBS262) cpcBAC HTPβ B64328(pBS168) cpcE^(c)inactivated by HTα transposon insertion B64328(pBS262) cpcE HTβB64407(pBS168) cpcF^(c) inactivated by HTα transposon insertionB64407(pBS262) cpcF HTβ PCC7120(pBS167) Wild-type HTα^(A12T)PCC7120(pBS162) Wild-type HTβ^(S46G,N76D) ^(a)The expression vectors,specified in parentheses, are derived from pBS150v and pBS150 asdescribed in the text. ^(b)Genes cpcB and cpcA encode the β and αsubunits of C-phycocyanin and cpcC encodes a linker polypeptide involvedin the assembly of trimers (αβ)₃ and hexamers [(αβ)₃]₂ and theirassembly into phycobilisomes (5,6). ^(c)cpcE and cpcF encode twosubunits of a heterodimeric phycocyanin α subunit lyase required for theaddition of phycocyanobilin to the apophycocyanin α subunit (1, 2-4).

TABLE 2 Molecular weights of His-tagged phycocyanins in differentaggregation states, as determined by size exclusion chromato- graphy andby analytical ultracentrifugation. Expected SEC-HPLC AUC Major Aggrega-MW Protein^(a) (kDa) (kDa) Components tion state (kDa) Native PC 211.7α:β Hexamer 225.7 124.7 Trimer 112.8 44.5 Monomer 37.6 An168 301.6 HTα:βHexamer 243.0 145.3 125.9 Trimer 121.5 49.2 34.8 Monomer 40.5 An262136.9 121.9 α:HTβ Trimer 121.5 62.4 43.4 Monomer 40.5 An168-BAC 289.3HTα:β Trimer 243.0 137.2 Monomer 121.5 17.3 HTα Subunit 20.9 monomerAn262-BAC 50.3 HTβ Subunit 45.0 homo- dimer An168-E/F 106.8 Apo- Trimer119.7 29.8 HTα:β Monomer 39.9 An262-E/F 54.1 HTβ Subunit 45.0 homo-dimer 33.8 Apo-α:HTβ Monomer 39.9 ^(a)“-BAC” suffix indicates proteinsisolated from cpcBAC mutant strains of Anabaena sp., and “-E” and “-F”indicate proteins isolated from cpcE and cpcF mutant strains of Anabaenasp., respectively.

TABLE 3 Spectroscopic properties of His-tagged phycocyanins^(a).Abs^(max) Ex^(max) Em^(max) εm (M⁻¹cm⁻¹) Sample (nm) (nm) (nm) (×1,000)Φ_(f) NativePC 615-618 615 644 929 ± 2.1 0.27 ± 0.06 (α:β)₃ (HTα:β)₃615-621 618 642 921 ± 36  0.22 ± 0.05 (α:HTB)₃ 615-619 616 642 888 ± 33 0.23 ± 0.04 HTα 618-622 615 637 109 ± 2.5 0.23 ± 0.02 (HTβ)₂ 605 606 639365 ± 13  0.22 ± 0.03 apo- 607 606 642 182 ± 1.4 0.24 ± 0.00 HTα:β One660^(b) — — 35.40 0 protein- bound 280 — — 14.85 ± 0.1 PCB^(a)Absorbance maxima (Abs^(max)) are for protein concentrations between0.05 and 3 μM. Lower concentrations yield blue-shifted spectra in allcases where a range of wavelengths is given. Excitation and emissionmaxima (Ex^(max) and Em^(max)), and quantum yields of fluorescence(Φ_(f)) are for samples at ≧0.05 μM. Molar extinction coefficients(ε_(m)) were determined for the following complexes # purified bySEC-HPLC :native PC, trimer fraction; HTα:β, fraction of An168; α:HTβ,trimer fraction of An262; HTα, subunit monomer fraction of An168-BAC;HTβ, subunit homodimer fraction of An262-BAC; apo-HTαβ: monomer fractionof An168-F. ^(b)Absorbance of protein-bound phycocyanobilin was measuredwith SEC-HPLC purified (HTα:β)₃ and (α:HTβ)₃ holoproteins, denatured in8 M urea pH 2.0. The molar extinction coefficient at 660 nm is identicalto that measured in 7.2 M urea pH 2.0, 9 mM DTT (35), while that at 280mn was calculated by subtracting contributions from Tyr [ε = 1,370 M⁻¹cm⁻¹; (36)] and # Trp ε = 5,500 M⁻¹ cm⁻¹; (37)]residues.

TABLE 4 Expression plasmids with specific functional domains in theencoded protein Coiled Affinity Biospec. coil Protease Core Plasmid^(a)tag tag^(b) domain site protein pBS311 6xHis GCN4pII TEV LacZ^(α) pBS3146xHis GCN4pII TEV CpcA pBS319 6xHis TEV CpcA-L9-CpcB pBS320 6xHis TEVCpcA-L11-CpcB pBS362 6xHis GCN4pII TEV CpcA-L11-CpcB pBS315 6xHis TEVCpcB-L11-CpcA pBS358 6xHis GCN4pII TEV CpcB-L11-CpcA pBS283 Strep2 TEVLacZ^(α) pBS342 6xHis Strep2 TEV LacZ^(α) pBS327 6xHis Strep2 TEV CpcApBS303 Strep2 GCN4pLI TEV LacZ^(α) pBS309 6xHis Strep2 GCN4pLI TEVLacZ^(α) pBS323 6xHis Strep2 GCN4pLI TEV CpcA pBS312 6xHis GCN4pLI TEVLacZ^(α) pBS321 6xHis GCN4pLI TEV CpcA ^(a)Like the parent plasmidspBS150v and pBS150, all plasmids listed have two versions: the smallerone, indicated in the text with a suffix “c”, is more suitable forcloning manipulations, but is lacking the 3.7-kb pDU1HC fragment thatenables autonomous plasmid replication in Anabaena sp. PCC7120 (ExampleA, herein). ^(b)The biospecificity tag is positioned on thecarboxyl-terminal side of the 6xHis tag, so that any recombinantmolecules that have lost the biospecificity tag through proteolysis arenot retained on the IMAC column. The Strep2 tag is a 10-residue peptidewith specific affinity for streptavidin.

TABLE 5 Determination of apparent molecular weight of components inrecombinant phycocyanin preparations by SEC-HPLC^(a) Calc. mass PercentAssembly Theoretical Protein (kDa) area state^(b) mass (kDa)^(c) An168 49.2 5 Monomer 40.5 (HTα:β) 145.3 95 Trimer 121.5 An327  44.2 4 Monomer41.4 (HT-Strep2-α:β) 137.9 94 Trimer 124.3 275.4 2 Hexamer 248.7 An314110.2 79 Trimer 132.7 (HT-pII-α:β) 406.0⁴ 21 (Hexamer)₂ 530.7 An321232.8 13 Tetramer 176.9 (HT-pLI-α:β) 499.5^(d) 81 (Tetramer)₃ 530.7An323 264.0 6 Tetramer 182.7 (HT-Strep2- 429.3^(d) 94 (Tetramer)₃ 548.2pLI-α:β) An319 358 79 Monomer 41.0 (HTα-L9-β)  99.3 19 Trimer 122.9232.6 2 Hexamer 245.8 An320  34.1 49 Monomer 41.1 (HTβ-L11-β)  89.7 46Trimer 123.4 213.6 5 Hexamer 246.7 An362 128.9 84 Trimer 134.5(HT-pII-α-L11-β) 304.7^(d) 15 Hexamer 269.1 An315 107.9 76 Trimer 123.4(HTβ-L11-α) 235.7 23 Hexamer 246.7 An358 115.6 64 Trimer 134.5(HT-pII-β-L11-α) 587.2^(d) 35 (Hexamer)₂ 538.0 ^(a)For experimentalconditions, see text. ^(b)By convention, the α:β heterodimer is referredto as a phycocyanin “monomer.” ^(c)Values are for holo-proteins.^(d)Apparent molecular weight outside the range defined by thecalibration standards.

TABLE 6 Spectroscopic properties of phycocyanin constructs^(a) Initialλ_(max) ^(c) A_(max)/ Ex_(max) Em_(max) ε_(M) Sample^(b) assembly state(nm) A_(360 nm) (nm) (nm) (M⁻¹cm⁻¹) Φ_(f) Denatured PC α:β 280 — — —  44,550^(d) 660 1.0 — —   106,200 0 Native PC (α:β)₃ 615-619 6.4 615644   929,000 0.27 An168 (HTα:β)₃ 615-621 6.4 618 642   921,000 0.22An327 (HT-Strep2- 615-619 6.4 618 642   922,000 0.23 α:β)₃ An262(α:HTβ)₃ 615-619 6.3 616 642   888,000 0.23 An314 (HT-pII-α:β)₃ 621-6227.5 622 642   927,000 0.39 An321 (HT-pLI- 621-623 7.5 621 643 1,231,0000.28 α:β)₄ An323 (HT-Strep2- 621-623 7.2 621 644 1,240,000 0.27pLI-α:β)₄ An315 (HTβ-L11-α)₃ 615-622 6.2 622 643   900,000 0.21 An358(HT-pII-β- 619-621 6.7 620 643   915,000 0.29 L11-α)₃ An320 (HTα-L11-β)₃605-608 5.2 613 643   677,000^(e) 0.22 An362 (HT-pII-α- 607-610 5.8 615642   707,000^(f) 0.24 L11-β)₃ ^(a)Trimer or tetramer componentsobtained by SEC-HPLC were used for all measurements. A_(max):A_(360 nm)values, excitation maxima (Ex_(max)), emission maxima (Em_(max)), andquantum yields of fluorescence (Φ_(f)) are given for measurements atprotein concentrations ≦0.05 mM. ε_(M) values are given for measurementson the components specified in column 2 at protein concentrations >1 μM.^(b)Properties of proteins not described in this study are taken fromExample A, herein. ^(c)λ_(max) values were determined at proteinconcentrations from 0.05 to 3 μM. Where the spectra shift to the bluewith decreasing protein concentration, the Iimits of λ_(max) values arethose measured at the lowest and highest concentrations, respectively.^(d)Absorbance from the three protein-bound PCBs only, excludingcontribution from Tyr and Trp residues (Example A, herein).

TABLE 7 List of fusion constructs indicating order of domains Fusionconstruct^(a) Plasmid^(b) 6XHis-BTN-TEV-LacZ^(α′) pBS3396xHis-BTN-TEV-CpcA pBS329 6xHis-BTN-pII-TEV-CpcA pBS3176xHis-BTN-TEV-GlbN pBS355 6xHis-TEV-GlbN pBS121 6xHis-TEV-TBN-BCCP114pBS344 6xHis-TEV-CpcB-TBN-BCCP114 pBS3536xHis-GCN4pII-TEV-CpcB-TBN-BCCP114 pBS3596xHis-TEV-CpcA-L11-CpcB-TBN-BCCP114 pBS3616xHis-GCN4pII-TEV-CpcA-L11-CpcB-TBN-BCCP114 pBS3656xHis-TEV-CpcA-TBN-BCCP114 pBS351 6xHis-GCN4pII-TEV-CpcA-TBN-BCCP114pBS357 6xHis-TEV-CpcB-L11-CpcA-TBN-BCCP114 pBS3606xHis-GCN4pII-TEV-CpcB-L11-CpcA-TBN-BCCP114 pBS364 6xHis-TEV-StreppBS370 6xHis-TEV-CpcA-Strep pBS386 6xHis-TEV-CpcB-L11-CpcA-Strep pBS394^(a)Functional domains in each construct are listed in order from N- toC-terminus, with particularly relevant domains in bold face. CpcA andCpcB correspond to the α and β subunits of phycocyanin, respectively.^(b)All plasmids have two versions. The smaller one, indicated in thetext with a suffix “v”, is for cloning and expression in E. coli, andlacks the 3.7-kb pDU1HC fragment that enables autonomous plasmidreplication in Anabaena sp. PCC7120 (Example A).

TABLE 8 Spectroscopic properties of phycocyanin constructs^(a) Initialassembly λ_(max) A_(max)/ Ex_(max) Em_(max) Sample state^(b) (nm)^(c)A_(360 nm) (nm) (nm) ε_(M) (M⁻¹ cm⁻¹) Φ_(f) Denatured α:β 280 — — — 44,550^(d) 0 PC 660 1.0 — — 106,200 0 Native (α:β)₃ 615-619 6.4 615 644929,000 0.27 PC An168 (HTα:β)₃ 615-621 6.4 618 642 921,000 0.22 An329(HT-BTN-α:β)₃ 615-618 6.4 617 642 920,000 0.22 An386 HTα-Strep:β 605-6095.6 607 639 186,000 0.19 An262 (α:HTβ)₃ 615-619 6.3 616 642 888,000 0.23An353 (α:HTβ- 611-616 5.7 612 642 896,000 0.21 BCCP¹¹⁴)₃ An314(HT-pII-α:β)₃ 621-622 7.5 622 642 927,000 0.39 An317 (HT-BTN-pII-621-622 7.3 622 644 939,000 0.31 α:β)₃ An359 (α:HT-pII-β- 619-622 6.6621 643 915,000 0.29 BCCP¹¹⁴)₃ An320 (HTα-L11-β)₃ 605-608 5.2 613 643677,000^(e) 0.22 An361 (HTα-L11-β- 605-606 5.3 604 637 664,000^(e) 0.23BCCP¹¹⁴)₃ An362 (HT-pII-α-L11- 607-610 5.8 615 642 707,000^(f) 0.24 β)₃An365 (HT-pII-α-L11- 605-607 5.5 606 641 692,000^(f) 0.27 β-BCCP¹¹⁴)₃^(a)Properties of constructs not described in this studies are takenfrom the accompanying studies (Example A, Example B).^(b)Affinity-purified proteins were further purified by SEC-HPLC, andcollected fractions in 50 mM Na-phosphate pH 7.0 buffer ± 1 mM NaN₃ wereused for all measurements. A_(max):A_(360nm) values, excitation maxima(Ex_(max)), emission maxima (Em_(max)), and quantum yields offluorescence (Φ_(f)) are given for measurements at proteinconcentrations ≦0.05 μM. ε_(M) values are given for measurements on thecomponents specified in column # 2 at protein concentrations >1 μM.^(c)λ_(max) values were determined at protein concentrations from 0.05to 3 μM. Where the spectra shift to the blue with decreasing proteinconcentration, the λ_(max) values are those measured at the lowest andhighest concentrations, respectively. ^(d)Absorbance from the threeprotein-bound PCBs only, corrected for contributions from Tyr and Trpresidues (Example A). ^(e)The phycocyanin α subunit domain has anaverage PCB content of ˜0.5. ^(f)The phycocyanin α subunit domain has anaverage PCB content of ˜0.25.

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FURTHER EXAMPLES 1. Phycobiliproteins Carrying the GCN4-pII or theGCN4-pLI Coiled-coil Oligomerization Domain

These fusion proteins were found almost entirely in inclusion bodies inE. coli, but found incorporated in the phycobilisome in Anabaena sp.These constructs, as well as other examples discussed below, all showthat proteins bearing the GCN4 oligomerization domains are found almostentirely in inclusion bodies when expressed in E. coli, but areincorporated in phycobilisomes when expressed in Anabaena sp. Uponphycobilisome dissociation, good yields of soluble, fluorescently taggedproteins are obtained.

2. Streptavidin-phycocyanin α Subunit Fusion Proteins

The core streptavidin (stvC) gene used here encodes a StvC correspondingto residues 16 to 133 of the mature streptavidin. Recombinant StvC witha 24-residue N-terminal extension bearing a 6×His tag and the TEV site,HT-StvC (encoded by plasmid pBS282), was expressed relatively poorly inE. coli, with about 80% of the proteins found in inclusion bodies. TheStvC-CpcA fusion protein has an 11-residue bridge linking the twomoieties. HT-StvC-CpcA (encoded by plasmid pBS292) was produced atrelatively high level in E. coli, but was found only in inclusionbodies.

The fusion protein An292 was well expressed in Anabaena sp.Ni²⁺-NTA-purified soluble An292 protein accounted for >15% of totalcellular phycobiliproteins. The An292 protein was of HT-StvC-α:β incomposition, ran on SEC-HPLC as tetramer (HT-StvC-α:β)4 and trimer oftetramers [(HT-StvC-α:β)4]3, similar to the finding with An321 withphycocyanin α subunit bearing the tetramerization domain GCN4-pLI:(HT-pLI-α:β)4 and [(HT-pLI-α:β)4]3. An292 also had spectroscopicproperties virtually identical to those of An321, making it an excellentfluorescent label. The An292 protein was assembled into phycobilisomesand also found to bind biotin.

Like streptavidin tetramers, the (HT-StvC-α:β)4 tetramer was much morestable than the (HT-pLI-α:β)4 tetramer. On room temperature denaturingSDS-PAGE, (HT-StvC-α:β)4 released only the β subunits and retained the(HT-StvC-α)4 aggregate. The tetramer could be broken only by boiling theprotein before loading on SDS-PAGE. Purified phycobilisomes displayingStvC had a tendency to aggregate out of solution, likely a direct resultof inter-phycobilisome linkage mediated by tetramerization of themonomeric StvC domains displayed on PBS. Usually within 48 hrs all suchphycobilisomes precipitated out of solution.

3. Protein A-phycocyanin α Subunit Fusion Proteins

Expression of truncated protein A (SpA) in the cytosol of E. coli hasbeen shown to give very poor yield, with particularly severe proteolysisoccuring to the hinge of domains A and B. In our construct, thetruncated SpA protein [denoted SpA(DABC)] contains the hinge of domainE, domains D, A, and B in entirety, and domain C lacking a small portionof its hinge. Domains D, A, B and C all retain their IgG-bindingstructure. Plasmid pBS356 encodes the recombinant SpA with a C-terminal6×His tag extending from the residual hinge region of domain C:SpA(DABC)-6×His. The protein, Ec356, was obtained in extremely pooryield when expressed in E. coli, regardless whether the purificationmethod was denaturing (using guanidine HCl; see e.g., Example B, supra)or not, and almost no full-length Ec356 protein was seen on SDS-PAGE.Recombinant protein A with engineered hinge regions has been describedfor better expression of the SpA in E.coli. Without such extensivereengineering, E. coli is not an appropriate organism for expression andproduction of recombinant protein A.

In experiments described here, the SpA(DABC) protein is fused at itsC-terminus to the phycocyanin a subunit through a 24-residue linkerbearing a 6×His tag and a recognition and cleavage site for the specificTEV endoprotease. The fusion protein, SpA(DABC)-6×His-TEV-CpcA, encodedby plasmid pBS349, was obtained in very poor yield when expressed inE.coli, likely a result of extensive proteolysis. Little full-lengthEc349 protein was seen, with most of the Ni2+-NTA-purified proteinhaving some or all of the IgG-binding domains missing.

When expressed in Anabaena sp., the fusion protein An349 was foundassembled into phycobilisomes with no apparent destabilizing effect onthe light-harvesting complex. Cellular phycobiliprotein level relativeto chlorophyll a, however, was about 20% lower, suggesting an elevatedphycobiliprotein turnover. Nonetheless, growth rate of cultures underhigh-light intensity was not affected. Ni2+-NTA-purified An349 proteinswere obtained in relatively low yield of about 5% of total cellularphycobiliproteins.

On SEC-HPLC, the An349 proteins fractionated into two components: >80%as the trimer (SpA-6×His-α:β)3 and the rest being SpA-6×His-α subunit.SDS-PAGE showed that the SpA moiety of the majority of the fusionprotein had also been proteolyzed, giving full-length SpA(DABC)-6×His-α(49.5 kDal; ca. 10%), SpA(ABC)-6×His-α (41.5 kDal; ca. 20%), andSpA(BC)-6×His-α (35.5 kDal; ca. 70%). No proteolytic cleavage on domainB hinge [giving SpA(C)-6×His-α] and domain C hinge (giving 6×His-α) wasobserved. The lack of proteolytic cleavage on the domain B hinge is aclear indication of different protease activities in Anabaena sp.because that hinge region has been found to be the most susceptible toproteolytic cleavage in E. coli. The lack of clipping in the domain Chinge is likely due to its proximity to the phycobilisome, minimizingaccess by proteases. This hinge region is susceptible to proteolyticactivities in Anabaena sp. when displayed farther away from thephycobilisome surface (see below).

SDS-PAGE also showed that the carrier phycocyanin a domain had thenormal bilin content. The SEC-HPLC trimer (SpA-6×His-α:β)3 fraction hadspectroscopic properties very similar to those of (6×His-α:β)3, withabsorbance maximum at 616 nm, an A616 nm:A360 nm ratio of 6.1,fluorescence excitation maximum at 618 nm (for 650 nm emission), andfluorescence emission maximum at 641 nm (for 560 nm excitation).

An349 proteins were tested for their ability to bind IgG in ELISA.Serially diluted An349 protein was immobilized on a 96-well plate andthen allowed to bind to mouse IgG-alkaline phosphatase conjugate. Afterthorough washing, the alkaline phosphatase activity was assayed bycatalyzed color development. In such semi-quantitative assays, theSpA-6×His-α:β fusion protein had about 30% IgG-binding activity ascompared to commercially obtained protein A (Sigma Chemical Co.) withall five IgG-binding domains.

Another SpA-phycocyanin a fusion protein was constructed to enhance thefusion protein's utility. This construct is similar to theSpA(DABC)-6×His-TEV-CpcA construct described above, but has the33-residue GCN4-pII trimerization domain inserted between the 6×His tagand the TEV site. This fusion protein, SpA(DABC)-6×His-pII-TEV-CpcA,encoded by plasmid pBS354, was found only in inclusion bodies whenexpressed in E. coli. Unlike Ec349, about 10% of the Ec354 proteinsaffinity-purified from inclusion bodies was full-length[SpA(DABC)-6×His-pII-TEV-CpcA], suggesting fast sequestering of thenewly synthesized polypeptides into inclusion bodies due to bundling bythe GCN4-pII domain. The majority of purified Ec354 proteins, however,were still missing some or all of the SpA IgG-binding domains, againillustrating protein A's susceptibility to E. coli proteases.

When expressed in Anabaena sp., the fusion protein An354 was foundassembled into phycobilisomes with no apparent destabilizing effect onthe complex. Interestingly, cells expressing An354 had a more normalphenotype than those expressing An349 (see above). Ni2+-NTA-purifiedAn354 proteins were obtained in reasonable yield, accounting for >7% oftotal cellular phycobiliproteins.

On SEC-HPLC, the An354 protein ran as a single peak with an apparentmolecular weight corresponding to a hexamer, [(SpA-6×His-pII-α:β)3]2.SDS-PAGE confirmed the composition as SpA-6×His-pII-α:β, and showed thatmost of the SpA moiety was subject to proteolytic clipping at differenthinge regions, giving full-length SpA(DABC)-6×His-pII-α (53.2 kDal; ca.10%), SpA(ABC)-6×His-pII-α (45.2 kDal; ca. 20%), SpA(BC)-6×His-pII-α(39.2 kDal; ca. 50%), and 6×His-pII-α (25.2 kDal; ca. 20%). The completeremoval of the displayed SpA protein by cleavage in the domain C hingeis not seen with the An349 protein, and is likely a result of the SpAmoiety in An354 being displayed farther away from the phycobilisomesurface. As with An349, no proteolytic cleavage on domain B hinge[giving SpA(C)-6×His-pII-α] was observed in Anabaena sp. Differentfractions collected from the An354 SEC-HPLC peak had identicaldistributions of the fusion proteins missing various IgG-bindingdomains, consistent with the view that the fusion proteins are displayedon the phycobilisome as monomers (with respect to the GCN4-pII domain),and only form GCN4-pII-bundled stable trimers after cell lysis andphycobilisome dissociation.

SDS-PAGE also showed that the carrier phycocyanin a domain had thenormal level of phycocyanobilin. The SEC-HPLC hexamer[(SpA-6×His-pII-α:β)3]2 fraction had spectroscopic properties verysimilar to those of (6×His-pII-α:β)3 (see, e.g. Example C, supra), withan absorbance maximum at 621 nm, an A616 nm:A360 nm ratio of 7.5,fluorescence excitation maximum at 621 nm (for 650 nm emission), andfluorescence emission maximum at 642 nm (for 560 nm excitation). Thesefavorable spectroscopic properties of such GCN4-pII-bundled stabletrimers make the fusion protein a good fluorescent tag. ELISA alsoshowed that An354 had IgG-binding affinity comparable to commerciallyobtained protein A. The increased IgG-binding activity of An354 in ELISAcompared to An349 may be a result of more protein being immobilized onthe 96-well plate, and/or cooperative binding of GCN4-pII-bundled SpAproteins. Such oligomerization-induced binding affinity increase hasbeen observed (see e.g., Examples B and C, supra).

4. Cyanoglobin Fusion to Phycocyanin α or β Subunit

Cyanoglobin (GlbN) is a 15.7-kDal monomeric hemoprotein produced in thecyanobacterium Nostoc commune. This protein contains a non-covalentlylinked heme, and is well expressed as soluble protein in both E. coli(expression at 30° C. and 37° C.) and Anabaena sp. when bearing a24-residue N-terminal extension containing a 6×His tag and the TEVrecognition and cleavage site (recombinant protein encoded by plasmidpBS121; Example C, supra). However, recombinant cyanoglobin withC-terminal extension (the stop codon is replaced by a Ser codon) wasfound unable to fold normally in E. coli. Cyanoglobin-phycocyaninsubunit fusion constructs, GlbN-6×His-TEV-CpcA (encoded by plasmidpBS190) and GlbN-6×His-TEV-CpcB (encoded by plasmid pBS274), gaverelatively poor yield and were found mostly in inclusion bodies whenexpression was carried out at 37° C. At 30° C., the two fusion proteinshad somewhat different behavior. Although both were still fast degradedin E. coli (thus giving low yield), the GlbN-HTa fusion protein (Ec190)was produced in a substantial amount as soluble protein, with apparentlynormal amount of heme and an absorbance spectrum identical to that of6×His-tagged GlbN (Ec121). The GlbN-HTb fusion protein (Ec274), on theother hand, was still found mostly in inclusion bodies, with the smallamount of soluble proteins purified largely as apoproteins (withoutheme). These results indicate that the C-terminal extensions have slowedthe folding of GlbN in E. coli, and the cyanoglobin domain in theGlbN-HTb fusion protein appears to be misfolded.

Anabaena cultures expressing either fusion proteins were healthy,showing no negative phenotypes at all. Both fusion proteins, An190 andAn274, were found assembled into phycobilisomes with no destabilizingeffect on the macrostructures, were purified in high yield (eachaccounting for >20% of total cellular phycobiliproteins) with cognatepartner subunits in 1:1 ratio, and found to have spectroscopicproperties very similar to native phycocyanin.

The cyanoglobin moiety in the fusion proteins, however, behaved quitedifferently. Using phycocyanin a subunit as the carrier domain (An190),the displayed GlbN domain was <30% holo when eluted off the Ni2+-NTAcolumn, and lost almost all of the bound heme upon dialysis to removeimidazole. Using the phycocyanin β subunit as the carrier domain(An274), most of the displayed GlbN domain was purified with bound heme,and the heme remained bound to GlbN after dialysis. No degradation ofthe cyanoglobin domain was observed in either An190 or An274 in Anabaenacells, suggesting that the cyanoglobin domain displayed on the N′ ofphycocyanin α subunit was misfolded but remained resistant toproteolysis. The misfolded GlbN domain appears to interfere with thecarrier domain's formation of phycocyanin trimers: on SEC-HPLC, about80% of An190 proteins ran as trimers, (GblN-HT α:β)3, while the rest ranas monomers, GblN-HT α:β in contrast to An168 which runs only astrimers, (HT α:β)3. Like An262, (α:HTβ)3, An274 ran only as trimers,(α:GlbN-HTα)3, on SEC-HPLC.

In sum, both fusion proteins were quickly degraded in E. coli, but welldisplayed on phycobilisomes in Anabaena sp. Cyanoglobin displayed on theof phycocyanin β subunit seemed to fold better than on the N-terminus ofthe α subunit, and was able to retain larger amount of heme than thelatter during purification. No degradation of the cyanoglobin domain wasobserved in either construct in Anabaena cells, suggesting that thecyanoglobin domain displayed on the N-terminus of phycocyanin α subunitwas misfolded but remained resistant to proteolysis.

5. Sperm Whale Myoglobin Fusion to Phycocyanin α or β Subunit

The myoglobin from sperm whale (SWMβ) is similar to cyanoglobin as amonomeric protein with non-covalently bound heme. SWMβ with a C-terminalextension of only six His residues (SWMβ-6×His; encoded by plasmidpBS114) can be well expressed as soluble holoprotein in both E. coli andAnabaena sp. Recombinant SWMβ proteins with larger C-terminalextensions, however, appear to be unable to fold.SWMβ-6×His-TEV-LacZ^(α) (encoded by plasmid pBS261), SWMβ-6×His-TEV-CpcA(encoded by plasmid pBS271), and SWMb-6×His-TEV-CpcB (encoded by plasmidpBS318) fusion proteins all gave poor yields when expressed in E. coliand were found nearly entirely in inclusion bodies.

Anabaena cultures expressing either An271 (SWMβ-HTα) or An361 (SWMβ-HTβ)fusion proteins were healthy, and the fusion proteins were foundassembled into phycobilisomes, and were purified with cognate partnerphycocyanin subunits. Although myoglobin displayed on the N-terminus ofphycocyanin β subunit (SWMβ-HTβ) gave slightly better yield of the fulllength protein (with little amount of bound heme), both fusion proteinswere purified mostly without the myoglobin moiety. This indicates thatthe displayed domain, if unable to fold, is degraded.

6. Maltose-binding Protein (MalE, MBP) Fusion to Phycocyanin α or βSubunit

The “mature” recombinant MalE protein (lacking the membrane-crossingsignal peptide) with a C-terminal 6×His tag is well expressed as asoluble protein in the cytosol of both Anabaena sp. and E. coli. In theMalE-phycocyanin fusion constructs, the last three residues(Arg-Ile-Thr) of the mature MalE are replaced by the spacer Asn-Ser-Ser,and this modified MalE is fused to the N-terminus of a phycocyaninsubunit via a 24-residue linker that bears a 6×His tag and a recognitionand cleavage site for the TEV protease. The MalE-6×His-TEV-α (Ec396) andMalE-6×His-TEV-β (Ec398) fusion proteins are both expressed as solubleproteins in E. coli in relatively high yield.

The strain Anabaena sp. PCC7120(pBS396) expressing fusion proteinMalE-6×His-TEV-α had a phenotype very similar to that of strain Anabaenasp. PCC7120(pBS168) expressing HTα. Sucrose density gradientsedimentation fractionation showed that the MalE-6×His-TEV-α fusionprotein was assembled into the phycobilisome without obvious effects onthe macromolecular complex. Ni²⁺-NTA affinity-purified protein, An396,was in relatively good yield, accounting for >10% of total cellularphycobiliproteins. Over 95% of the An396 proteins can bind to, and bespecifically eluted from amylose columns, diagnostic of functional MalE.

SDS-PAGE showed that the An396 protein has the compositionMalE-6×His-TEV-α:β, and Zn2+-induced fluorescence indicates fullchromophorylation of phycocyanin carrier domain. SEC-HPLC showed thatAn396 protein was monomeric, (MalE-6×His-TEV-α:β) a behavior differingfrom that of HTα:β, whose elution volume is that of a trimer. TheMalE-6×His-TEV-α:β protein in the SEC-HPLC monomer peak fraction,however, has spectroscopic properties very similar to the (HTα:β)3trimer, with absorbance maximum at 617 nm, fluorescence excitationmaximum at 618 nm (for 650 nm emission), fluorescence emission max. at637 nm (for 560 nm excitation), and a fluorescence quantum yield of0.23.

Upon digestion with the TEV protease, over 90% of purifiedMalE-6×His-TEV-α:β protein can be clipped at the TEV recognition site toseparate the displayed domain (MalE) from the phycocyanin carrierdomain.

The fusion protein MalE-6×His-TEV-β is expressed in Anabaena sp. at alevel (>7.5% of total cellular phycobiliproteins) comparable to that ofMalE-6×His-TEV-α (An396). Over 95% of Ni2+-NTA-purified An398 proteincan bind to, and then be specifically eluted from, the amylose resin,indicative of the MalE function. However, analysis of An398 by SDS-PAGEshows that the protein preparation consists mostly of MalE-6×His-TEV-βwith only a very small amount of the partner phycocyanin α subunit.Zn2+-induced fluorescence suggests that the MalE-6×His-TEV-β protein hasa full complement of phycocyanobilin, i.e., two PCBs per β subunitdomain. On SEC-HPLC most of the preparation runs as (MalE-6×His-TEV-β)2homodimer, with a slight shoulder corresponding to the(α:MalE-6×His-TEV-β) monomer. Like MalE-6×His-TEV-α:β (An396), virtuallyall of the MalE-6×His-TEV-β fusion proteins can be cleaved at the TEVsite to separate the displayed protein (MalE) and the carrier protein(phycocyanin β subunit).

The MalE-6×His-TEV-β protein is found to be assembled intophycobilisomes without obvious deleterious effect on the latterstructures. Since assembly of the fusion protein into the phycobilisomesrequires association with the phycocyanin α subunit, this resultindicates that the MalE-6×His-TEV-β fusion protein has a reducedaffinity to the α subunit which is likely lost during the purificationprocess. In the absence of α subunits, phycocyanin β subunits have beenshown to form stable homodimers (see, e.g. Example A, supra). Suchinterference by the displayed domain on the phycocyanin subunit carrierdomain's affinity for its cognate partner subunit is likely fusionprotein specific—cyanoglobin displayed on the N-terminus of thephycocyanin β subunit does not appear to have obvious effects on thefusion protein's affinity for the phycocyanin α subunit.

The (MalE-6×His-TEV-β)2 homodimer peak fraction in SEC-HPLC was used forspectroscopic characterization. Similar to the (HTβ)2 homodimer protein,the (MalE-6×His-TEV-β)2 homodimer has absorbance and fluorescenceexcitation maxima at 605 nm, a fluorescence emission maximum at 638 nm,and a fluorescence quantum yield of 0.26.

To obtain 1:1 α:β stoichiometry, the β-L11-α subunit-fusion phycocyanin(Example B, supra) can be used, in place of the β subunit, as a carrierprotein. Constructs incorporating the GCN4-pII trimerization domain,such as (MalE-6×His-pII-α:β)3 and (MalE-6×His-pII-β-L11-α)3, haveexcellent spectroscopic properties, and are very useful, for instance,in studies of protein glycosylation.

All publications and patent applications cited in this specification andall references cited therein are herein incorporated by reference as ifeach individual publication or patent application or reference werespecifically and individually indicated to be incorporated by reference.Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to those of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

What is claimed is:
 1. A fusion protein comprising a functionaldisplayed domain and a functional phycobiliprotein domain incorporatedin a functional oligomeric phycobiliprotein.
 2. The fusion protein ofclaim 1 wherein the phycobiliprotein domain is a naturalphycobiliprotein domain.
 3. The fusion protein of claim 1 wherein thefunctional oligomeric phycobiliprotein is an α,β heterodimer.
 4. Thefusion protein of claim 1 wherein the displayed domain comprises amoiety selected from the group consisting of an affinity tag, anoligomerization moiety, a specific binding moiety, and a signalingmoiety.
 5. The fusion protein of claim 1 further comprising a specificbinding moiety selected from a streptavidin biotin-binding moiety, abiotinylated or biotinylatable moiety, and an antigen bindingimmunoglobulin moiety.
 6. The fusion protein of claim 1 furthercomprising a linker peptide between the displayed domain and thephycobiliprotein domain.
 7. The fusion protein of claim 1 furthercomprising a protease cleavage site between the displayed domain and thephycobiliprotein domain.
 8. The fusion protein of claim 1 wherein thephycobiliprotein domain comprises at least one functional attachedbilin.
 9. The fusion protein of claim 1 wherein the displayed domain isrefractive to expression in E. coli.
 10. A functional phycobilisomecomprising the fusion protein of claim
 1. 11. A method for making afusion protein, comprising a functional displayed domain and afunctional phycobiliprotein domain incorporated in a functionaloligomeric phycobiliprotein, the method comprising the steps of:providing a nucleic acid encoding a polypeptide comprising a functionaldisplayed domain and a functional phycobiliprotein domain; making thepolypeptide by expressing the nucleic acid in a cell or cell-freeexpression system; and combining the polypeptide with a phycobiliproteinsubunit under conditions to form the fusion protein.
 12. A method forisolating a functional displayed domain, the method comprising the stepsof: making the fusion protein according to the method of claim 11; afterthe combining step, cleaving a peptide bond between the functionaldisplayed domain and the functional phycobiliprotein domain; andseparating the functional displayed domain from the functionalphycobiliprotein domain.
 13. The method of claim 11, wherein the makingand combining steps occur in a cell.
 14. The method of claim 11, whereinthe making and combining steps occur in a cell, and the cell is or is aprogeny of a cell which naturally expresses a phycobiliprotein.
 15. Themethod of claim 14, wherein the fusion protein further comprises afunctionally attached bilin.
 16. The method of claim 14, wherein thefission protein further comprises a linker peptide between the displayeddomain and the phycobiliprotein domain.
 17. The method of claim 14,wherein the fusion protein further comprises a protease cleavage sitebetween the displayed domain and the phycobiliprotein domain.
 18. Themethod of claim 14, wherein the displayed domain is refractive toexpression in E. coli.